T cell activating bispecific antigen binding molecules

ABSTRACT

The present invention generally relates to novel bispecific antigen binding molecules for T cell activation and re-direction to specific target cells. In addition, the present invention relates to polynucleotides encoding such bispecific antigen binding molecules, and vectors and host cells comprising such polynucleotides. The invention further relates to methods for producing the bispecific antigen binding molecules of the invention, and to methods of using these bispecific antigen binding molecules in the treatment of disease.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically in ASCII format and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Nov. 12, 2015, isnamed P32186-US-Seq-Listing.txt and is 445,556 bytes in size.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims priority to European Patent Application No.14194147.6, filed on Nov. 20, 2014, the entire contents of which areincorporated herein by reference.

FIELD OF THE INVENTION

The present invention generally relates to bispecific antigen bindingmolecules for activating T cells, in particular to bispecific antibodiestargeting CD3 and Folate Receptor 1 (FolR1). In addition, the presentinvention relates to polynucleotides encoding such bispecific antigenbinding molecules, and vectors and host cells comprising suchpolynucleotides. The invention further relates to methods for producingthe bispecific antigen binding molecules of the invention, and tomethods of using these bispecific antigen binding molecules in thetreatment of disease.

BACKGROUND

The selective destruction of an individual cell or a specific cell typeis often desirable in a variety of clinical settings. For example, it isa primary goal of cancer therapy to specifically destroy tumor cells,while leaving healthy cells and tissues intact and undamaged.

An attractive way of achieving this is by inducing an immune responseagainst the tumor, to make immune effector cells such as natural killer(NK) cells or cytotoxic T lymphocytes (CTLs) attack and destroy tumorcells. CTLs constitute the most potent effector cells of the immunesystem, however they cannot be activated by the effector mechanismmediated by the Fc domain of conventional therapeutic antibodies.

In this regard, bispecific antibodies designed to bind with one “arm” toa surface antigen on target cells, and with the second “arm” to anactivating, invariant component of the T cell receptor (TCR) complex,have become of interest in recent years. The simultaneous binding ofsuch an antibody to both of its targets will force a temporaryinteraction between target cell and T cell, causing activation of anycytotoxic T cell and subsequent lysis of the target cell. Hence, theimmune response is re-directed to the target cells and is independent ofpeptide antigen presentation by the target cell or the specificity ofthe T cell as would be relevant for normal MHC-restricted activation ofCTLs. In this context it is crucial that CTLs are only activated when atarget cell is presenting the bispecific antibody to them, i.e. theimmunological synapse is mimicked. Particularly desirable are bispecificantibodies that do not require lymphocyte preconditioning orco-stimulation in order to elicit efficient lysis of target cells.

FOLR1 is expressed on epithelial tumor cells of various origins, e.g.,ovarian cancer, lung cancer, breast cancer, renal cancer, colorectalcancer, endometrial cancer. Several approaches to target FOLR1 withtherapeutic antibodies, such as farletuzumab, antibody drug conjugates,or adoptive T cell therapy for imaging of tumors have been described(Kandalaft et al., J Transl Med. 2012 Aug. 3; 10:157. doi:10.1186/1479-5876-10-157; van Dam et al., Nat Med. 2011 Sep. 18;17(10):1315-9. doi: 10.1038/nm.2472; Clifton et al., Hum Vaccin. 2011February; 7(2):183-90. Epub 2011 Feb. 1; Kelemen et al., Int J Cancer.2006 Jul. 15; 119(2):243-50; Vaitilingam et al., J Nucl Med. 2012 July;53(7); Teng et al., 2012 August; 9(8):901-8. doi:10.1517/17425247.2012.694863. Epub 2012 Jun. 5. Some attempts have beenmade to target folate receptor-positive tumors with constructs thattarget the folate receptor and CD3 (Kranz et al., Proc Natl Acad SciUSA. Sep. 26, 1995; 92(20): 9057-9061; Roy et al., Adv Drug Deliv Rev.2004 Apr. 29; 56(8):1219-31; Huiting Cui et al Biol Chem. Aug. 17, 2012;287(34): 28206-28214; Lamers et al., Int. J. Cancer. 60(4):450 (1995);Thompson et al., MAbs. 2009 July-August; 1(4):348-56. Epub 2009 Jul. 19;Mezzanzanca et al., Int. J. Cancer, 41, 609-615 (1988). However, theapproaches taken so far have many disadvantages. The molecules used thusfar could not be readily and reliably produced as they require chemicalcross linking. Similarly, hybrid molecules cannot be produced at largescale as human proteins and require the use of rat, murine or otherproteins that are highly immunogenic when administered to humans and,thus, of limited therapeutic value. Further, many of the existingmolecules retained FcgR binding.

Thus, there remains a need for novel, improved bispecific antibodies fortargeted T cell mediated immunotherapy. The present invention providesbispecific antigen binding molecules designed for targeted T cellactivation, particularly, bispecific antigen binding molecules suitableas effective and safe therapeutics that can be readily produced anddosed.

SUMMARY OF THE INVENTION

In one aspect, the invention provides a T cell activating bispecificantigen binding molecule comprising

-   -   (i) a first antigen binding moiety which is a Fab molecule        capable of specific binding to CD3, and which comprises at least        one heavy chain complementarity determining region (CDR) amino        acid sequence selected from the group consisting of SEQ ID NO:        37, SEQ ID NO: 38 and SEQ ID NO: 39 and at least one light chain        CDR selected from the group of SEQ ID NO: 32, SEQ ID NO: 33, SEQ        ID NO: 34; and    -   (ii) a second antigen binding moiety capable of specific binding        to Folate Receptor 1 (FolR1).

In one embodiment, the T cell activating bispecific antigen bindingmolecule comprises a first antigen binding moiety that comprises avariable heavy chain comprising an amino acid sequence of SEQ ID NO: 36and a variable light chain comprising an amino acid sequence of SEQ IDNO: 31. In one embodiment, the T cell activating bispecific antigenbinding molecule additionally comprises (iii) a third antigen bindingmoiety capable of specific binding to FolR1. In one embodiment, thesecond and third antigen binding moiety capable of specific binding toFolR1 comprise identical heavy chain complementarity determining region(CDR) and light chain CDR sequences. In one embodiment, the thirdantigen binding moiety is identical to the second antigen bindingmoiety. In one embodiment of the T cell activating bispecific antigenbinding molecule of the above embodiments, at least one of the secondand third antigen binding moiety is a Fab molecule. In one embodiment,the T cell activating bispecific antigen binding molecule of the aboveembodiments, additionally comprises an Fc domain composed of a first anda second subunit capable of stable association. In some embodiments, thefirst antigen binding moiety and the second antigen binding moiety areeach connected at the C-terminus of the Fab heavy chain to theN-terminus of the first or second subunit of the Fc domain. In someembodiments, a third antigen binding moiety is fused at the C-terminusof the Fab heavy chain to the N-terminus of the Fab heavy chain of thefirst antigen binding moiety, optionally via a peptide linker.

In one embodiment of the T cell activating bispecific antigen bindingmolecule of the above embodiments, the antigen binding moiety capable ofspecific binding to Folate Receptor 1 (FolR1) comprises at least oneheavy chain complementarity determining region (CDR) amino acid sequenceselected from the group consisting of SEQ ID NO: 16, SEQ ID NO: 17 andSEQ ID NO: 18 and at least one light chain CDR selected from the groupof SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34. In one embodiment, theantigen binding moiety capable of specific binding to Folate Receptor 1(FolR1) comprises a variable heavy chain comprising an amino acidsequence of SEQ ID NO: 15 and a variable light chain comprising an aminoacid sequence of SEQ ID NO: 31. In one embodiment, the antigen bindingmoiety capable of specific binding to Folate Receptor 1 (FolR1)comprises at least one heavy chain complementarity determining region(CDR) amino acid sequence selected from the group consisting of SEQ IDNO: 8, SEQ ID NO: 56 and SEQ ID NO: 57 and at least one light chain CDRselected from the group of SEQ ID NO: 59, SEQ ID NO: 60, SEQ ID NO: 65.In one embodiment, the antigen binding moiety capable of specificbinding to Folate Receptor 1 (FolR1) comprises a variable heavy chaincomprising an amino acid sequence of SEQ ID NO: 55 and a variable lightchain comprising an amino acid sequence of SEQ ID NO: 64.

In another embodiment, the antigen binding moiety capable of specificbinding to Folate Receptor 1 (FolR1) comprises at least one heavy chaincomplementarity determining region (CDR) amino acid sequence selectedfrom the group consisting of SEQ ID NO: 8, SEQ ID NO: 9 and SEQ ID NO:50 and at least one light chain CDR selected from the group of SEQ IDNO: 52, SEQ ID NO: 53, SEQ ID NO: 54. In one embodiment, the antigenbinding moiety capable of specific binding to FolR1 comprises (a) acomplementarity determining region heavy chain 1 (CDR-H1) amino acidsequences of SEQ ID NO: 8; (b) a CDR-H2 amino acid sequence of SEQ IDNO: 9; (c) a CDR-H3 amino acid sequence of SEQ ID NO: 50; (d) acomplementarity determining region light chain 1 (CDR-L1) amino acidsequence of SEQ ID NO: 52; (e) a CDR-L2 amino acid sequence of SEQ IDNO: 53, and (f) a CDR-L3 amino acid sequence of SEQ ID NO: 54. In oneembodiment, the antigen binding moiety capable of specific binding toFolR1 comprises a variable heavy chain comprising an amino acid sequenceof SEQ ID NO: 49 and a variable light chain comprising an amino acidsequence of SEQ ID NO: 51.

In another embodiment, the antigen binding moiety capable of specificbinding to Folate Receptor 1 (FolR1) comprises at least one heavy chaincomplementarity determining region (CDR) amino acid sequence selectedfrom the group consisting of SEQ ID NO: 16, SEQ ID NO: 275 and SEQ IDNO: 315 and at least one light chain CDR selected from the group of SEQID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34. In one embodiment, the antigenbinding moiety capable of specific binding to FolR1 comprises (a) acomplementarity determining region heavy chain 1 (CDR-H1) amino acidsequences of SEQ ID NO: 16; (b) a CDR-H2 amino acid sequence of SEQ IDNO: 275; (c) a CDR-H3 amino acid sequence of SEQ ID NO: 315; (d) acomplementarity determining region light chain 1 (CDR-L1) amino acidsequence of SEQ ID NO: 32; (e) a CDR-L2 amino acid sequence of SEQ IDNO: 33, and (f) a CDR-L3 amino acid sequence of SEQ ID NO: 34. In oneembodiment, the antigen binding moiety capable of specific binding toFolR1 comprises a variable heavy chain domain (VH) comprising an aminoacid sequence of SEQ ID NO: 274 and a variable light chain domain (VL)comprising an amino acid sequence of SEQ ID NO: 31.

In one embodiment, the T cell activating bispecific antigen bindingmolecule of the above embodiments binds to a human FolR1. In oneembodiment, the T cell activating bispecific antigen binding molecule ofthe above embodiments binds to a human FolR1 and a cynomolgus monkeyFolR1. In one embodiment, the T cell activating bispecific antigenbinding molecule of the above embodiments binds to a human FolR1, acynomolgus monkey FolR1 and a murine FolR1. In one embodiment, the Tcell activating bispecific antigen binding molecule of the aboveembodiments binds to a human FolR1, a cynomolgus monkey FolR1 and not amurine FolR1.

In one embodiment of the T cell activating bispecific antigen bindingmolecule of any of the above embodiments, the first antigen bindingmoiety is a crossover Fab molecule wherein either the variable or theconstant regions of the Fab light chain and the Fab heavy chain areexchanged. In one embodiment, the T cell activating bispecific antigenbinding molecule of any of the above embodiments comprises not more thanone antigen binding moiety capable of specific binding to CD3. In oneembodiment of the T cell activating bispecific antigen binding molecule,the first and the second antigen binding moiety and the Fc domain arepart of an immunoglobulin molecule. In one embodiment, the Fc domain isan IgG class immunoglobulin, specifically an IgG₁ or IgG₄, Fc domain. Inone embodiment, the Fc domain is a human Fc domain.

In one embodiment of the T cell activating bispecific antigen bindingmolecule of any of the above embodiments, the Fc domain comprises amodification promoting the association of the first and the secondsubunit of the Fc domain. In one embodiment, in the CH3 domain of thefirst subunit of the Fc domain an amino acid residue is replaced with anamino acid residue having a larger side chain volume, thereby generatinga protuberance within the CH3 domain of the first subunit which ispositionable in a cavity within the CH3 domain of the second subunit,and in the CH3 domain of the second subunit of the Fc domain an aminoacid residue is replaced with an amino acid residue having a smallerside chain volume, thereby generating a cavity within the CH3 domain ofthe second subunit within which the protuberance within the CH3 domainof the first subunit is positionable. In one embodiment, the Fc domaincomprises at least one amino acid substitution that reduces binding toan Fc receptor and/or effector function, as compared to a native IgG₁ Fcdomain. In one embodiment, each subunit of the Fc domain comprises threeamino acid substitutions that reduce binding to an activating Fcreceptor and/or effector function wherein said amino acid substitutionsare L234A, L235A and P329G (Kabat numbering). In one embodiment, the Fcreceptor is an Fcγ receptor. In one embodiment, the effector function isantibody-dependent cell-mediated cytotoxicity (ADCC).

In one embodiment of the T cell activating bispecific antigen bindingmolecule of any of the above embodiments, the T cell activatingbispecific antigen binding molecule induces proliferation of a human CD3positive T cell in vitro. In one embodiment, the T cell activatingbispecific antigen binding molecule induces human peripheral bloodmononuclear cell mediated killing of a FolR1-expressing human tumor cellin vitro. In one embodiment, the T cell activating bispecific antigenbinding molecule induces T cell mediated killing of a FolR1-expressinghuman tumor cell in vitro. In one embodiment, the T cell is a CD8positive T cell. In one embodiment, the FolR1-expressing human tumorcell is a Hela, Skov-3, HT-29, or HRCEpiC cell. In one embodiment, the Tcell activating bispecific antigen binding molecule induces T cellmediated killing of the FolR1-expressing human tumor cell in vitro withan EC50 of between about 36 pM and about 39573 pM after 24 hours. In oneembodiment, the T cell activating bispecific antigen binding moleculeinduces T cell mediated killing of the FolR1-expressing tumor cell invitro with an EC50 of about 36 pM after 24 hours. In one embodiment, theT cell activating bispecific antigen binding molecule induces T cellmediated killing of the FolR1-expressing tumor cell in vitro with anEC50 of about 178.4 pM after 24 hours. In one embodiment, the T cellactivating bispecific antigen binding molecule induces T cell mediatedkilling of the FolR1-expressing tumor cell in vitro with an EC50 ofabout 134.5 pM or greater after 48 hours.

In one embodiment, the T cell activating bispecific antigen bindingmolecule of any of the above embodiments induces upregulation of cellsurface expression of at least one of CD25 and CD69 on the T cell asmeasured by flow cytometry. In one embodiment, the T cell is a CD4positive T cell or a CD8 positive T cell. In one embodiment, the T cellactivating bispecific antigen binding molecule of any of the aboveembodiments binds human FolR1 with an apparent K_(D) of about 5.36 pM toabout 4 nM. In one embodiment, the T cell activating bispecific antigenbinding molecule binds human and cynomolgus FolR1 with an apparent K_(D)of about 4 nM. In one embodiment, the T cell activating bispecificantigen binding molecule binds murine FolR1 with an apparent K_(D) ofabout 1.5 nM. In one embodiment, the T cell activating bispecificantigen binding molecule binds human FolR1 with a monovalent bindingK_(D) of at least about 1000 nM.

In one embodiment, the T cell activating bispecific antigen bindingmolecule of any of the above embodiments is specific for FolR1 and doesnot bind to FolR2 or FolR3. In one embodiment, the T cell activatingbispecific antigen binding molecule of any of the above embodiments hasan affinity (monovalent binding) of 1 μM or greater. In one embodiment,the affinity is around 1.4 μM for human FolR1. In one embodiment, the Tcell activating bispecific antigen binding molecule of any of the aboveembodiments has an avidity (bivalent binding) of about 1-100 nM orlower. In one embodiment, the avidity is about 10 nM or less. In oneembodiment, the avidity is 10 nM.

In one embodiment, the T cell activating bispecific antigen bindingmolecule of any of the above embodiments binds to FolR1 expressed on ahuman tumor cell. In one embodiment, the T cell activating bispecificantigen binding molecule of any of the above embodiments binds to aconformational epitope on human FolR1. In one embodiment, the T cellactivating bispecific antigen binding molecule of any of the aboveembodiments does not bind to human Folate Receptor 2 (FolR2) or to humanFolate Receptor 3 (FolR3). In one embodiment of the T cell activatingbispecific antigen binding molecule of any of the above embodiments, theantigen binding moiety binds to a FolR1 polypeptide comprising the aminoacids 25 to 234 of human FolR1 (SEQ ID NO:227). In one embodiment of theT cell activating bispecific antigen binding molecule of any of theabove embodiments, the FolR1 antigen binding moiety binds to a FolR1polypeptide comprising the amino acid sequence of SEQ ID NOs:227, 230and 231, and wherein the FolR1 antigen binding moiety does not bind to aFolR polypeptide comprising the amino acid sequence of SEQ ID NOs:228and 229.

In another aspect, the invention provides for a bispecific antibodycomprising a) a first antigen-binding site that competes for binding tohuman FolR1 with a reference antibody comprising a variable heavy chaindomain (VH) of SEQ ID NO: 49 and a variable light chain domain of SEQ IDNO: 51; and b) a second antigen-binding site that competes for bindingto human CD3 with a reference antibody comprising a variable heavy chaindomain (VH) of SEQ ID NO: 36 and a variable light chain domain of SEQ IDNO: 31, wherein binding competition is measured using a surface plasmonresonance assay.

In another aspect, the invention provides for a bispecific antibodycomprising a) a first antigen-binding site that competes for binding tohuman FolR1 with a reference antibody comprising a variable heavy chaindomain (VH) of SEQ ID NO: 274 and a variable light chain domain of SEQID NO: 31; and b) a second antigen-binding site that competes forbinding to human CD3 with a reference antibody comprising a variableheavy chain domain (VH) of SEQ ID NO: 36 and a variable light chaindomain of SEQ ID NO: 31, wherein binding competition is measured using asurface plasmon resonance assay.

In another aspect, the invention provides for a T cell activatingbispecific antigen binding molecule comprising a first antigen bindingmoiety capable of specific binding to CD3, and a second antigen bindingmoiety capable of specific binding to Folate Receptor 1 (FolR1), whereinthe T cell activating bispecific antigen binding molecule binds to thesame epitope on human FolR1 as a first reference antibody comprising avariable heavy chain domain (VH) of SEQ ID NO: 49 and a variable lightchain domain of SEQ ID NO: 51; and wherein the T cell activatingbispecific antigen binding molecule binds to the same epitope on humanCD3 as a second reference antibody comprising a variable heavy chaindomain (VH) of SEQ ID NO: 36 and a variable light chain domain of SEQ IDNO: 31.

In another aspect, the invention provides for a T cell activatingbispecific antigen binding molecule comprising a first antigen bindingmoiety capable of specific binding to CD3, and a second antigen bindingmoiety capable of specific binding to Folate Receptor 1 (FolR1), whereinthe T cell activating bispecific antigen binding molecule binds to thesame epitope on human FolR1 as a first reference antibody comprising avariable heavy chain domain (VH) of SEQ ID NO: 274 and a variable lightchain domain (VL) of SEQ ID NO: 31; and wherein the T cell activatingbispecific antigen binding molecule binds to the same epitope on humanCD3 as a second reference antibody comprising a variable heavy chaindomain (VH) of SEQ ID NO: 36 and a variable light chain domain (VL) ofSEQ ID NO: 31.

In another aspect, the invention relates to an antibody or anantigen-binding fragment thereof that competes for binding to humanFolR1 with an antibody that comprises a variable heavy chain domain (VH)of SEQ ID NO: 274 and a variable light chain domain of SEQ ID NO: 31,wherein binding competition is measured using a surface plasmonresonance assay.

In one aspect, the invention provides for a T cell activating bispecificantigen binding molecule, wherein the antigen binding molecule comprisesa first, second, third, fourth and fifth polypeptide chain that form afirst, a second and a third antigen binding moiety, wherein the firstantigen binding moiety is capable of binding CD3 and the second and thethird antigen binding moiety each are capable of binding Folate Receptor1 (FolR1), wherein a) the first and the second polypeptide chaincomprise, in amino (N)-terminal to carboxyl (C)-terminal direction, VLD1and CLD1; b) the third polypeptide chain comprises, in N-terminal toC-terminal direction, VLD2 and CH1D2; c) the fourth polypeptide chaincomprises, in N-terminal to C-terminal direction, VHD1, CH1D1, CH2D1 andCH3D1; d) the fifth polypeptide chain comprises VHD1, CH1D1, VHD2, CLD2,CH2D2 and CH3D2; wherein VLD1 is a first light chain variable domain,VLD2 is a second light chain variable domain, CLD1 is a first lightchain constant domain, CLD2 is a second light chain constant domain,VHD1 is a first heavy chain variable domain, VHD2 is a second heavychain variable domain, CH1D1 is a first heavy chain constant domain 1,CH1D2 is a second heavy chain constant domain 1, CH2D1 is a first heavychain constant domain 2, CH2D2 is a second heavy chain constant domain2, CH3D1 is a first heavy chain constant domain 3, and CH3D2 is a secondheavy chain constant domain 3.

In one embodiment of the T cell activating bispecific antigen bindingmolecule, (i) the third polypeptide chain and VHD2 and CLD2 of the fifthpolypeptide chain form the first antigen binding moiety capable ofbinding CD3; (ii) the first polypeptide chain and VHD1 and CH1D1 of thefourth polypeptide chain form the second binding moiety capable ofbinding to FolR1; and (iii) the second polypeptide chain and VHD1 andCH1D1 of the fifth polypeptide chain form the third binding moietycapable of binding to FolR1. In one embodiment, CH2D1, CH3D1, CH2D2 andCH3D2 form an Fc domain of an IgG class immunoglobulin. In oneembodiment, the Fc domain is a human Fc domain. In one embodiment, theFc domain comprises a modification promoting the association of thefirst and the second subunit of the Fc domain. In one embodiment, CH3D2comprises an amino acid residue having a larger side chain volume, whichis positionable in a cavity within CH3D1. In one embodiment, the Fcdomain comprises at least one amino acid substitution that reducesbinding to an Fc receptor and/or effector function, as compared to anative IgG₁ Fc domain. In one embodiment, each subunit of the Fc domaincomprises three amino acid substitutions that reduce at least one ofbinding to an activating Fc receptor and effector function wherein saidamino acid substitutions are L234A, L235A and P329G according to Kabatnumbering. In one embodiment, the Fc receptor is an Fcγ receptor. In oneof the above embodiments, the T cell activating bispecific antigenbinding molecule induces proliferation of a human CD3 positive T cell invitro. In one of the above embodiments, the T cell activating bispecificantigen binding molecule induces human peripheral blood mononuclear cellmediated killing of a FolR1-expressing human tumor cell in vitro. In oneof the above embodiments, the T cell activating bispecific antigenbinding molecule induces T cell mediated killing of a FolR1-expressinghuman tumor cell in vitro. In one such embodiment, the FolR1-expressinghuman tumor cell is a Hela, Skov-3, HT-29, or HRCEpiC cell. In one ofthe above embodiments, the T cell activating bispecific antigen bindingmolecule induces T cell mediated killing of the FolR1-expressing tumorcell in vitro with an EC50 of between about 36 pM and about 39573 pMafter 24 hours. In one of the above embodiments, the T cell activatingbispecific antigen binding molecule induces T cell mediated killing ofthe FolR1-expressing tumor cell in vitro with an EC50 of about 36 pMafter 24 hours. In one of the above embodiments, the T cell activatingbispecific antigen binding molecule induces T cell mediated killing ofthe FolR1-expressing tumor cell in vitro with an EC50 of about 178.4 pMafter 24 hours. In one of the above embodiments, the T cell activatingbispecific antigen binding molecule induces T cell mediated killing ofthe FolR1-expressing tumor cell in vitro with an EC50 of about 134.5 pMor greater after 48 hours. In one of the above embodiments, the T cellactivating bispecific antigen binding molecule induces upregulation ofcell surface expression of at least one of CD25 and CD69 on the T cellas measured by flow cytometry. In one such embodiments, the T cell is aCD4 positive T cell or a CD8 positive T cell. In one of the aboveembodiments, wherein the T cell activating bispecific antigen bindingmolecule binds human FolR1 with an apparent K_(D) of about 5.36 pM toabout 4 nM. In one of the above embodiments, the T cell activatingbispecific antigen binding molecule binds human and cynomolgus FolR1with an apparent K_(D) of about 4 nM. In one of the above embodiments,the T cell activating bispecific antigen binding molecule binds murineFolR1 with an apparent K_(D) of about 1.5 nM. In one of the aboveembodiments, the T cell activating bispecific antigen binding moleculebinds human FolR1 with a monovalent binding K_(D) of at least about 1000nM. In one of the above embodiments, the T cell activating bispecificantigen binding molecule binds to FolR1 expressed on a human tumor cell.In one of the above embodiments, the T cell activating bispecificantigen binding molecule binds to a conformational epitope on humanFolR1. In one of the above embodiments, the T cell activating bispecificantigen binding molecule does not bind to human Folate Receptor 2(FolR2) or to human Folate Receptor 3 (FolR3). In one of the aboveembodiments, the antigen binding moiety binds to a FolR1 polypeptidecomprising the amino acids 25 to 234 of human FolR1 (SEQ ID NO:227). Inone of the above embodiments, the FolR1 antigen binding moiety binds toa FolR1 polypeptide comprising the amino acid sequence of SEQ IDNOs:227, 230 and 231, and wherein the FolR1 antigen binding moiety doesnot bind to a FolR polypeptide comprising the amino acid sequence of SEQID NOs:228 and 229. In one of the above embodiments, the T cellactivating bispecific antigen binding molecule is a humanized or achimeric molecule. In one of the above embodiments, VHD2 and CH1D1 arelinked through a peptide linker.

In one of the above embodiments of the T cell activating bispecificantigen binding molecule, the first and second polypeptide chaincomprise the amino acid sequence of SEQ ID NO:230. In one of the aboveembodiments of the T cell activating bispecific antigen bindingmolecule, the third polypeptide chain comprises the amino acid sequenceof SEQ ID NO:86. In one of the above embodiments of the T cellactivating bispecific antigen binding molecule, the fourth polypeptidechain comprises the amino acid sequence of SEQ ID NO:309. In one of theabove embodiments of the T cell activating bispecific antigen bindingmolecule, the fifth polypeptide chain comprises the amino acid sequenceof SEQ ID NO:308. In one of the above embodiments of the T cellactivating bispecific antigen binding molecule, the first and secondpolypeptide chain comprise the amino acid sequence of SEQ ID NO:230; thethird polypeptide chain comprises the amino acid sequence of SEQ IDNO:86; the fourth polypeptide chain comprises the amino acid sequence ofSEQ ID NO:309; and the fifth polypeptide chain comprise the amino acidsequence of SEQ ID NO:308.

In one aspect, the invention provides for a T cell activating bispecificantigen binding molecule comprising the amino acid sequence of SEQ IDNO:308. In one embodiment, the T cell activating bispecific antigenbinding molecule of the above embodiment further comprises the aminoacid sequence of SEQ ID NO:230 and of SEQ ID NO:86.

In one aspect, the invention provides for an isolated polypeptidecomprising the amino acid sequence of SEQ ID NO:308. In one aspect, theinvention provides for an isolated polypeptide comprising the amino acidsequence of SEQ ID NO:309.

In one aspect, the invention provides for a T cell activating bispecificantigen binding molecule comprising the amino acid sequence of SEQ IDNO:276. In one embodiment, the T cell activating bispecific antigenbinding molecule of the above embodiment further comprises the aminoacid sequence of SEQ ID NO:277 and of SEQ ID NO:35.

In one aspect, the invention provides for an isolated polypeptidecomprising the amino acid sequence of SEQ ID NO:277. In one aspect, theinvention provides for an isolated polypeptide comprising the amino acidsequence of SEQ ID NO:276.

In one aspect, the invention provides for an isolated polynucleotideencoding the T cell activating bispecific antigen binding molecule ofany one of the embodiments disclosed herein. In one embodiment, theinvention provides for an isolated polynucleotide encoding a T cellactivating bispecific antigen binding molecule comprising the nucleotidesequence of SEQ ID NO:169. In one embodiment, the invention provides foran isolated polynucleotide encoding a T cell activating bispecificantigen binding molecule comprising the nucleotide sequence of SEQ IDNO:246. In one embodiment, the invention provides for an isolatedpolynucleotide encoding a T cell activating bispecific antigen bindingmolecule comprising the nucleotide sequence of SEQ ID NO:247. In oneembodiment, the invention provides for an isolated polynucleotideencoding a T cell activating bispecific antigen binding moleculecomprising the nucleotide sequence of SEQ ID NO:97. In one embodiment,the invention provides for an isolated polynucleotide encoding a T cellactivating bispecific antigen binding molecule comprising the nucleotidesequence of SEQ ID NO:198.

In one embodiment, the invention provides for an isolated polynucleotideencoding a T cell activating bispecific antigen binding moleculecomprising the nucleotide sequence of SEQ ID NO:287. In one embodiment,the invention provides for an isolated polynucleotide encoding a T cellactivating bispecific antigen binding molecule comprising the nucleotidesequence of SEQ ID NO:288. In one embodiment, the invention provides foran isolated polynucleotide encoding a T cell activating bispecificantigen binding molecule comprising the nucleotide sequence of SEQ IDNO:289.

In one aspect, the invention provides for an isolated polypeptideencoded by the polynucleotide of the above embodiment. In anotheraspect, the invention provides for a vector, particularly an expressionvector, comprising the polynucleotide encoding the T cell activatingbispecific antigen binding molecule of any one of the embodimentsdisclosed herein. In another aspect, the invention provides for a hostcell comprising a polynucleotide or a vector of any of the embodimentsdisclosed herein.

In one aspect, the invention provides for a method of producing the Tcell activating bispecific antigen binding molecule capable of specificbinding to CD3 and a target cell antigen, comprising the steps of a)culturing the host cell of the above embodiments under conditionssuitable for the expression of the T cell activating bispecific antigenbinding molecule and b) recovering the T cell activating bispecificantigen binding molecule.

In one aspect, the invention provides for T cell activating bispecificantigen binding molecule produced by the method of the above embodiment.

In one aspect, the invention provides for a pharmaceutical compositioncomprising the T cell activating bispecific antigen binding molecule ofany one of the above embodiments and a pharmaceutically acceptablecarrier. In one aspect, the invention provides for the T cell activatingbispecific antigen binding molecule of any one of the above embodimentsor the pharmaceutical composition of any of the above embodiments foruse as a medicament.

In one aspect, the invention provides for the T cell activatingbispecific antigen binding molecule of any one of the above embodimentsor the pharmaceutical composition of any one of the above embodimentsfor use in the treatment of a disease in an individual in need thereof.In some embodiments, the disease is cancer. In one aspect, the inventionprovides for a use of the T cell activating bispecific antigen bindingmolecule of any one of the above embodiments for the manufacture of amedicament for the treatment of a disease in an individual in needthereof.

In one aspect, the invention provides for a method of treating a diseasein an individual, comprising administering to said individual atherapeutically effective amount of a composition comprising the T cellactivating bispecific antigen binding molecule of any one of the aboveembodiments in a pharmaceutically acceptable form. In some embodiments,said disease is a cancer.

In one aspect, the invention provides for a method for inducing lysis ofa target cell, comprising contacting a target cell with the T cellactivating bispecific antigen binding molecule of any one of the aboveembodiments in the presence of a T cell.

In one aspect, the invention provides for a the invention as describedhereinbefore.

BRIEF DESCRIPTION OF THE DRAWINGS

The patent or application file contains at least one drawing executed incolor. Copies of this patent or patent application publication withcolor drawing(s) will be provided by the Office upon request and paymentof the necessary fee.

FIGS. 1A-I illustrate exemplary configurations of the T cell activatingbispecific antigen binding molecules (TCBs) of the invention. Allconstructs except the kappa-lambda format in (FIG. 1I) have P329G LALAmutations and comprise knob-into-hole Fc fragments with knob-into-holemodifications. (FIG. 1A) Illustration of the “FolR1 TCB 2+1 inverted(common light chain)”. The FolR1 binder is fused at the C-terminus ofthe Fab heavy chain to the N-terminus of the first subunit of the Fcdomain comprising the knob modification. These constructs are notcrossed and have three times the same VLCL light chain. (FIG. 1B)Illustration of the “FolR1 TCB 1+1 head-to-tail (common light chain)”.These constructs are not crossed and have two times the same VLCL lightchain. (FIG. 1C) Illustration of the “FolR1 TCB 1+1 classical (commonlight chain)”. These constructs are not crossed and have two times thesame VLCL light chain. (FIG. 1D) Illustration of the “FolR1 TCB 2+1classical (common light chain)”. The CD3 binder is fused at theC-terminus of the Fab heavy chain to the N-terminus of the first subunitof the Fc domain comprising the knob modification. These constructs arenot crossed and have three times the same VLCL light chain. (FIG. 1E)Illustration of the “FolR1 TCB 2+1 crossfab classical”. These constructscomprise a Ck-VH chain for the CD3 binder instead of the conventionalCH1-VH chain. The CD3 binder is fused at the C-terminus of the Fab heavychain to the N-terminus of the first subunit of the Fc domain comprisingthe knob modification. (FIG. 1F) Illustration of the “FolR1 TCB 2+1crossfab inverted”. These constructs comprise a Ck-VH chain for the CD3binder instead of the conventional CH1-VH chain. The FolR1 binder isfused at the C-terminus of the Fab heavy chain to the N-terminus of thefirst subunit of the Fc domain comprising the knob modification. (FIG.1G) Illustration of the “FolR1 TCB 1+1 crossfab head-to-tail”. Theseconstructs comprise a Ck-VH chain for the CD3 binder instead of theconventional CH1-VH chain. (FIG. 1H) Illustration of the “FolR1 TCB 1+1crossfab classical”. These constructs comprise a Ck-VH chain for the CD3binder instead of the conventional CH1-VH chain. FIG. 1I illustrates theCD3/FolR1 kappa-lambda antibody format. These constructs comprise acrossed common light chain VLCH1 and one crossed VHCL chain specific forCD3 and one crossed VHCL chain specific for FolR1.

FIGS. 2A-C depict graphs summarizing Binding of FoLR1 IgG binders toHeLa cells. Binding of newly generated FolR1 binders to FolR1 expressedon HeLa cells were determined by flow cytometry. Bound antibodies weredetected with a fluorescently labeled anti-human secondary antibody.

FIGS. 3A-B depict graphs summarizing specificity of FolR1 binders forFolR1. Binding of FolR1 IgGs to HEK cells transiently transfected witheither FolR1 or FolR2 was analyzed by flow cytometry to identify cloneswhich bind specifically to FolR1 and not to FolR2. The antibodies weredetected with a fluorescently labeled anti-human secondary antibody.

FIGS. 4A-B depict graphs summarizing cross-reactivity of FolR1 bindersto cyFoLR1. Cross-reactivity of the FolR1 antibodies to cyno FolR1 wasaddressed on HEK cells transiently transfected with cyFolR1 by flowcytometry. The antibodies were detected with a fluorescently labeledanti-human secondary antibody.

FIG. 5 depicts a graph illustrating internalization of FolR1 TCBs afterbinding. Internalization of the four FolR1 TCBs after binding to FolR1was tested on HeLa cells. Remaining FolR1 TCBs on the surface weredetected with a fluorescently labeled anti-human secondary antibodyafter indicated time points of incubation at 37° C. Percentage ofinternalization was calculated.

FIGS. 6A-E depict graphs summarizing binding of FolR1 IgGs to cells withdifferent FolR1 expression levels. Binding of 9D11, 16D5 and Mov19 IgGto tumor cells with different FolR1 expression levels was analyzed byflow cytometry. DP47 IgG was included as isotype control and MKN-45 wereincluded as FolR1 negative cell line. The antibodies were detected witha fluorescently labeled anti-human secondary antibody.

FIGS. 7A-L depict graphs summarizing T cell mediated killing of HT-29and SKOV3 cells. FolR1 TCBs were used to test T cell mediated killing ofHT-29 and SKOV3 tumor cells and upregulation of activation marker on Tcells upon killing. (FIGS. 7A-D) T cell mediated killing of HT-29 andSKOV3 cells in the presence of 9D11 FolR1 TCB and 16D5 FolR1 TCB wasmeasured by LDH release after 24 h and 48 h. DP47 TCB was included asnegative control. After 48 h incubation upregulation of the activationmarker CD25 and CD69 on CD8 T cells and CD4 T cells upon killing ofSKOV3 (FIGS. 7E-H) or HT-29 (FIG. 7I-L) tumor cells was assessed by flowcytometry.

FIG. 8 depicts a graph showing absence of anti-FolR1 binding toerythrocytes. Erythrocytes were gated as CD235a positive population andbinding of 9D11 IgG, 16D5 IgG, Mov19 IgG and DP47 IgG to this populationwas determined by flow cytometry. The antibodies were detected with afluorescently labeled anti-human secondary antibody.

FIGS. 9A-D depict graphs summarizing activation marker upregulation inwhole blood. CD25 and CD69 activation marker upregulation of CD4 T cellsand CD8 T cells 24 h after addition of 9D11 FolR1 TCB, 16D5 FolR1 TCB,Mov19 FolR1 TCB and DP47 TCB was analyzed by flow cytometry.

FIG. 10 Binding of 9D11 TCB a-glyco variants to HeLa cells. Binding of9D11 FolR1 TCB a-glyco variants to Hela cells was compared to binding ofthe original 9D11 TCB on HeLa cells. The antibodies were detected with afluorescently labeled anti-human secondary antibody and binding wasdetermined by flow cytometry.

FIGS. 11A-F depict graphs summarizing T cell mediated killing with 9D11FolR1 TCB a-glyco variants of tumor cells. 9D11 FolR1 TCB a-glycovariants were used to test T cell mediated killing of (FIG. 11A-D)SKOV3, MKN-45 (as FolR1 negative control) and (FIG. 11E-F) HT-29 tumorcells in comparison to killing with the original 9D11 FolR1 TCB. Asread-out LDH release after 24 h and 48 h was used.

FIGS. 12A-X depict graphs summarizing T cell mediated killing of primaryepithelial cells. Primary epithelial cells with very low levels of FolR1were used to test T cell mediated killing with 16D5 FolR1 TCB and 9D11FolR1 TCB, DP47 TCB was included as a negative control and HT29 cellswere included as positive control. (FIGS. 12A-H) LDH release of humanretinal pigment (HRP), human renal cortical (HRC), human bronchial (HB)and of HT29 cells was determined after 24 h and 48 h. CD25 and CD69activation marker upregulation on CD4 T cells and CD8 T cells uponkilling of (FIGS. 12I-L) HRP, (FIGS. 12M-P) HRC, (FIGS. 12Q-T) HB and(FIGS. 12 U-X) HT29 was determined after 48 h by flow cytometry.

FIGS. 13A-C show a comparison of different TCB formats with 16D5. Fourdifferent TCB formats containing the FolR1 binder 16D5 were compared inFIG. 13A binding to HeLa cells, in FIG. 14 B T cell mediated killing ofSKOV3 cells after 24 h and 48 h and in FIG. 14C CD25 and CD69 activationmarker upregulation on CD4 T cells and CD8 T cells 48 h after killing.

FIGS. 14A-C depict a comparison of different TCB formats with 9D11.Three different TCB formats containing the FolR1 binder 9D11 werecompared in A) binding to HeLa cells, in B) T cell mediated killing ofSKOV3 cells after 24 h and 48 h and in C) CD25 and CD69 activationmarker upregulation on CD4 T cells and CD8 T cells 48 h after killing.

FIG. 15 depicts a PK-profile of FOLR1 TCB in NOG mice for threedifferent doses.

FIG. 16 illustrates an experimental protocol for efficacy study withFOLR1 TCB.

FIGS. 17A-B depict tumor growth curves. (FIG. 17A) Mean values and SEMof tumor volumes in the different treatment groups. (FIG. 17B) Tumorgrowth of single mice in all treatment groups. TGI (tumor growthinhibition) give the percentage of the Mean tumor volume compared tovehicle group.

FIG. 18 shows tumor weights at study termination.

FIGS. 19A-B show FACS analysis of tumor infiltrating T-cells at studyday 32. (FIG. 19A) Tumor single cells suspensions were stained withanti-human CD3/CD4/CD8 and analyzed by flow cytometry. (FIG. 19B) Meanvalues and SEM of T-cell counts per mg tumor tissue in differenttreatment groups.

FIGS. 20A-B show FACS analysis for T-cell activation/degranulation andcytokine secretion at study day 32. CD4+(FIG. 20A) and CD8+(FIG. 20B)tumor infiltrating T-cells were stained for cytokines, activation anddegranulation markers. Displayed are the mean values and SEM of T-cellcounts per mg tumor tissue in different treatment groups.

FIGS. 21A-B show percent tumor lysis. SKOV3 cells were incubated withPBMCs in the presence of either kappa lambda FoLR1 TCB or DP47 TCB.After 24 h (FIG. 21A) and 48 h (FIG. 21B) killing of tumor cells wasdetermined by measuring LDH release.

FIGS. 22A-D show CD25 and CD69 upregulation on CD4 T cells. SKOV3 cellswere incubated with PBMCs in the presence of either kappa lambda FoLR1TCB or DP47 TCB. After 48 h CD25 and CD69 upregulation on CD4 T cells(FIG. 22A-B) and CD8 T cells (FIG. 22C-D) was measured by flowcytometry.

FIGS. 23A-B show percent tumor lysis. T-cell killing of SKov-3 cells(medium FolR1) induced by 36F2 TCB, Mov19 TCB and 21A5 TCB after 24 h(FIG. 23A) and 48 h (FIG. 23B) of incubation (E:T=10:1, effectors humanPBMCs).

FIGS. 24A-C show T-cell killing induced by 36F2 TCB, 16D5 TCB, 16D5 TCBclassical, 16D5 TCB 1+1 and 16D5 TCB HT of Hela (high FolR1) (FIG. 24A),Skov-3 (medium FolR1) (FIG. 24B) and HT-29 (low FolR1) (FIG. 24C) humantumor cells (E:T=10:1, effectors human PBMCs, incubation time 24 h).DP47 TCB was included as non-binding control.

FIGS. 25A-C show upregulation of CD25 and CD69 on human CD8+(FIG. 25A,B) and CD4+(FIG. 25C), T cells after T cell-mediated killing of Helacells (high FolR1) (FIG. 25A), SKov-3 cells (medium FolR1) (FIG. 25B)and HT-29 cells (low FolR1) (FIG. 25C) (E:T=10:1, 48 h incubation)induced by 36F2 TCB, 16D5 TCB and DP47 TCB (non-binding control).

FIGS. 26A-F show T-cell killing induced by 36F2 TCB, 16D5 TCB and DP47TCB of human Renal Cortical Epithelial Cells (FIG. 26A, B), humanRetinal Pigment Epithelial Cells (FIG. 26C, D) and HT-29 cells (FIG.26E, F) cells after 24 h (FIG. 26A, C, E) and 48 h (FIG. 26B, D, F) ofincubation (E:T=10:1, effectors human PBMCs).

FIG. 27 depicts a table summarizing quantification of FolR1 bindingsites on various normal and cancer cells lines.

FIGS. 28A-B show binding of 16D5 TCB and its corresponding CD3deamidation variants 16D5 TCB N100A and 16D5 TCB S100aA and 9D11 TCB andits demidation variants 9D11 TCB N100A and 9D11 TCB S100aA to human CD3expressed on Jurkat cells.

FIGS. 29A-B show T-cell killing of SKov-3 (medium FolR1) human tumorcells induced by 16D5 TCB and its corresponding CD3 deamidation variants16D5 TCB N100A and 16D5 TCB S100aA (FIG. 29A) and 9D11 TCB and itsdemidation variants 9D11 TCB N100A and 9D11 TCB S100aA (FIG. 29B)(E:T=10:1, effectors human PBMCs, incubation time 24 h). DP47 TCB wasincluded as non-binding control.

FIG. 30A-B show T-cell killing of HT-29 (low FolR1) human tumor cellsinduced by 16D5 TCB and its corresponding CD3 deamidation variants 16D5TCB N100A and 16D5 TCB S100aA (FIG. 30A) and 9D11 TCB and its demidationvariants 9D11 TCB N100A and 9D11 TCB S100aA (FIG. 30B) (E:T=10:1,effectors human PBMCs, incubation time 24 h). DP47 TCB was included asnon-binding control.

FIGS. 31A-C show mean fluorescence intensity and tumor cell lysis.

FIGS. 32A-E shows binding of 36F2 TCB, 16D5 TCB and 16D5 HC/LC variantsto human FolR1 expressed on Hela cells.

FIG. 33 shows binding of 36F2 TCB, 16D5 TCB and the two 16D5 affinityreduced variants 16D5 W96Y/D52E TCB and 16D5 G49S/S93A TCB to humanFolR1 on Hela cells.

FIGS. 34A-E show binding of 36F2 TCB, 16D5 TCB and 16D5 HC/LC variantsto human FolR1 expressed on HT-29 cells.

FIGS. 35A-D show binding of intermediate FolR1 binders (6E10 TCB, 14B1TCB and 9C7 TCB), 16D5 TCB and 36F2 TCB to HEK293T cells expressingeither human or mouse FolR1 or FolR2.

FIG. 36A-F show T-cell killing of Hela (high FolR1 expression), SKov-3(medium FolR1 expression) and HT-29 (low FolR1 expression) human tumorcells induced by intermediate FolR1 binders (6E10 TCB, 14B1 TCB and 9C7TCB), 16D5 TCB and 36F2 TCB after 24 h (A-C) and 48 h (D-F) ofincubation. Human PBMCs were used as effector cells (E:T=10:1).

FIG. 37A-F shows T-cell killing of Hela (high FolR1 expression), SKov-3(medium FolR1 expression) and HT-29 (low FolR1 expression) human tumorcells induced by affinity reduced 16D5 variants (16D5-G49S/S93A TCB,16D5-G49S/K53A TCB, 16D5 W96Y TCB, 16D5 W96Y/D52E TCB), 16D5 TCB and36F2 TCB after 24 h (FIG. 38A-C) and 48 h (FIG. 38D-F) of incubation.Human PBMCs were used as effector cells (E:T=10:1).

FIG. 38A-F show T-cell killing of primary human cells from retinalpigment epithelium and renal cortical epithelium induced by affinityreduced 16D5 variants (16D5-G49S/S93A TCB, 16D5 W96Y/D52E TCB), 16D5TCB, 36F2 TCB and the intermediate FolR1 binder 14B1 TCB was assessedafter 24 h (FIG. 39A-C) and 48 h (FIG. 39D-F) of incubation (E:T=10:1,effectors human PBMCs). HT-29 cells (low FolR1 expression) were includedas control cell line and DP47 TCB served as non-binding control.

FIGS. 39A-B show single dose PK of FOLR1 TCB constructs in female NOGmice.

FIGS. 40A-G show in vivo efficacy of FOLR1 TCB constructs (16D5, 16D5G49S/S93A and 16D5 W96Y/D52E) after human PBMC transfer in Hela-bearingNOG mice.

FIG. 41 shows that Farletuzumab (dark green, second from the top) andMov19 (grey, top) are able to bind on huFolR1 that is captured on 16D5,demonstrating that the 16D5 series binders recognize an epitope distinctfrom that recognized by either Farletuzumab or Mov19.

DETAILED DESCRIPTION OF THE INVENTION Definitions

Terms are used herein as generally used in the art, unless otherwisedefined in the following.

As used herein, the term “antigen binding molecule” refers in itsbroadest sense to a molecule that specifically binds an antigenicdeterminant. Examples of antigen binding molecules are immunoglobulinsand derivatives, e.g. fragments, thereof.

The term “bispecific” means that the antigen binding molecule is able tospecifically bind to at least two distinct antigenic determinants.Typically, a bispecific antigen binding molecule comprises at least twoantigen binding sites, each of which is specific for a differentantigenic determinant. In certain embodiments the bispecific antigenbinding molecule is capable of simultaneously binding two antigenicdeterminants, particularly two antigenic determinants expressed on twodistinct cells.

The term “valent” as used herein denotes the presence of a specifiednumber of antigen binding sites in an antigen binding molecule. As such,the term “monovalent binding to an antigen” denotes the presence of one(and not more than one) antigen binding site specific for the antigen inthe antigen binding molecule.

An “antigen binding site” refers to the site, i.e. one or more aminoacid residues, of an antigen binding molecule which provides interactionwith the antigen. For example, the antigen binding site of an antibodycomprises amino acid residues from the complementarity determiningregions (CDRs). A native immunoglobulin molecule typically has twoantigen binding sites, a Fab molecule typically has a single antigenbinding site.

As used herein, the term “antigen binding moiety” refers to apolypeptide molecule that specifically binds to an antigenicdeterminant. In one embodiment, an antigen binding moiety is able todirect the entity to which it is attached (e.g. a second antigen bindingmoiety) to a target site, for example to a specific type of tumor cellor tumor stroma bearing the antigenic determinant. In another embodimentan antigen binding moiety is able to activate signaling through itstarget antigen, for example a T cell receptor complex antigen. Antigenbinding moieties include antibodies and fragments thereof as furtherdefined herein. Particular antigen binding moieties include an antigenbinding domain of an antibody, comprising an antibody heavy chainvariable region and an antibody light chain variable region. In certainembodiments, the antigen binding moieties may comprise antibody constantregions as further defined herein and known in the art. Useful heavychain constant regions include any of the five isotypes: α, δ, ε, γ, orμ. Useful light chain constant regions include any of the two isotypes:κ and λ.

As used herein, the term “antigenic determinant” is synonymous with“antigen” and “epitope,” and refers to a site (e.g. a contiguous stretchof amino acids or a conformational configuration made up of differentregions of non-contiguous amino acids) on a polypeptide macromolecule towhich an antigen binding moiety binds, forming an antigen bindingmoiety-antigen complex. Useful antigenic determinants can be found, forexample, on the surfaces of tumor cells, on the surfaces ofvirus-infected cells, on the surfaces of other diseased cells, on thesurface of immune cells, free in blood serum, and/or in theextracellular matrix (ECM). The proteins referred to as antigens herein,e.g., FolR1 and CD3, can be any native form the proteins from anyvertebrate source, including mammals such as primates (e.g. humans) androdents (e.g. mice and rats), unless otherwise indicated. In aparticular embodiment the antigen is a human protein. Where reference ismade to a specific protein herein, the term encompasses the“full-length”, unprocessed protein as well as any form of the proteinthat results from processing in the cell. The term also encompassesnaturally occurring variants of the protein, e.g. splice variants orallelic variants. Exemplary human proteins useful as antigens include,but are not limited to: FolR1 (Folate receptor alpha (FRA); Folatebinding protein (FBP); human FolR1 UniProt no.: P15328; murine FolR1UniProt no.: P35846; cynomolgus FolR1 UniProt no.: G7PR14) and CD3,particularly the epsilon subunit of CD3 (see UniProt no. P07766 (version130), NCBI RefSeq no. NP_000724.1, SEQ ID NO:150 for the human sequence;or UniProt no. Q95LI5 (version 49), NCBI GenBank no. BAB71849.1, for thecynomolgus [Macaca fascicularis] sequence). The T cell activatingbispecific antigen binding molecule of the invention binds to an epitopeof CD3 or a target cell antigen that is conserved among the CD3 ortarget antigen from different species. In certain embodiments the T cellactivating bispecific antigen binding molecule of the invention binds toCD3 and FolR1, but does not bind to FolR2 (Folate receptor beta; FRB;human FolR2 UniProt no.: P14207) or FolR3 (Folate receptor gamma; humanFolR3 UniProt no.: P41439).

By “specific binding” is meant that the binding is selective for theantigen and can be discriminated from unwanted or non-specificinteractions. The ability of an antigen binding moiety to bind to aspecific antigenic determinant can be measured either through anenzyme-linked immunosorbent assay (ELISA) or other techniques familiarto one of skill in the art, e.g. surface plasmon resonance (SPR)technique (analyzed on a BIAcore instrument) (Liljeblad et al., Glyco J17, 323-329 (2000)), and traditional binding assays (Heeley, Endocr Res28, 217-229 (2002)). In one embodiment, the extent of binding of anantigen binding moiety to an unrelated protein is less than about 10% ofthe binding of the antigen binding moiety to the antigen as measured,e.g., by SPR. In certain embodiments, an antigen binding moiety thatbinds to the antigen, or an antigen binding molecule comprising thatantigen binding moiety, has a dissociation constant (K_(D)) of ≦1 μM,≦100 nM, ≦10 nM, ≦1 nM, ≦0.1 nM, ≦0.01 nM, or ≦0.001 nM (e.g. 10⁻⁸M orless, e.g. from 10⁻⁸M to 10⁻¹³M, e.g., from 10⁻⁹M to 10⁻¹³ M).

“Affinity” refers to the strength of the sum total of non-covalentinteractions between a single binding site of a molecule (e.g., areceptor) and its binding partner (e.g., a ligand). Unless indicatedotherwise, as used herein, “binding affinity” refers to intrinsicbinding affinity which reflects a 1:1 interaction between members of abinding pair (e.g., an antigen binding moiety and an antigen, or areceptor and its ligand). The affinity of a molecule X for its partner Ycan generally be represented by the dissociation constant (K_(D)), whichis the ratio of dissociation and association rate constants (k_(off) andk_(on), respectively). Thus, equivalent affinities may comprisedifferent rate constants, as long as the ratio of the rate constantsremains the same. Affinity can be measured by well-established methodsknown in the art, including those described herein. A particular methodfor measuring affinity is Surface Plasmon Resonance (SPR).

“Reduced binding”, for example reduced binding to an Fc receptor, refersto a decrease in affinity for the respective interaction, as measuredfor example by SPR. For clarity the term includes also reduction of theaffinity to zero (or below the detection limit of the analytic method),i.e. complete abolishment of the interaction. Conversely, “increasedbinding” refers to an increase in binding affinity for the respectiveinteraction.

“T cell activation” as used herein refers to one or more cellularresponse of a T lymphocyte, particularly a cytotoxic T lymphocyte,selected from: proliferation, differentiation, cytokine secretion,cytotoxic effector molecule release, cytotoxic activity, and expressionof activation markers. The T cell activating bispecific antigen bindingmolecules of the invention are capable of inducing T cell activation.Suitable assays to measure T cell activation are known in the artdescribed herein.

A “target cell antigen” as used herein refers to an antigenicdeterminant presented on the surface of a target cell, for example acell in a tumor such as a cancer cell or a cell of the tumor stroma. Inparticular “target cell antigen” refers to Folate Receptor 1.

As used herein, the terms “first” and “second” with respect to antigenbinding moieties etc., are used for convenience of distinguishing whenthere is more than one of each type of moiety. Use of these terms is notintended to confer a specific order or orientation of the T cellactivating bispecific antigen binding molecule unless explicitly sostated.

A “Fab molecule” refers to a protein consisting of the VH and CH1 domainof the heavy chain (the “Fab heavy chain”) and the VL and CL domain ofthe light chain (the “Fab light chain”) of an immunoglobulin.

The term “Fab molecules having identical VLCL light chains” as usedtherein refers to binders that share one light chain but still haveseparate specificities, e.g., can bind CD3 or FolR1. In some embodimentsthe T-cell activating bispecific molecules comprise at least two Fabmolecules having identical VLCL light chains. The corresponding heavychains are remodeled and confer specific binding to the T cellactivating bispecific antigen CD3 and the target cell antigen FolR1,respectively.

By “fused” is meant that the components (e.g. a Fab molecule and an Fcdomain subunit) are linked by peptide bonds, either directly or via oneor more peptide linkers.

As used herein, the term “single-chain” refers to a molecule comprisingamino acid monomers linearly linked by peptide bonds. In certainembodiments, one of the antigen binding moieties is a single-chain Fabmolecule, i.e. a Fab molecule wherein the Fab light chain and the Fabheavy chain are connected by a peptide linker to form a single peptidechain. In a particular such embodiment, the C-terminus of the Fab lightchain is connected to the N-terminus of the Fab heavy chain in thesingle-chain Fab molecule.

By a “crossover” Fab molecule (also termed “Crossfab”) is meant a Fabmolecule wherein either the variable regions or the constant regions ofthe Fab heavy and light chain are exchanged, i.e. the crossover Fabmolecule comprises a peptide chain composed of the light chain variableregion and the heavy chain constant region, and a peptide chain composedof the heavy chain variable region and the light chain constant region.For clarity, in a crossover Fab molecule wherein the variable regions ofthe Fab light chain and the Fab heavy chain are exchanged, the peptidechain comprising the heavy chain constant region is referred to hereinas the “heavy chain” of the crossover Fab molecule. Conversely, in acrossover Fab molecule wherein the constant regions of the Fab lightchain and the Fab heavy chain are exchanged, the peptide chaincomprising the heavy chain variable region is referred to herein as the“heavy chain” of the crossover Fab molecule. An antibody that comprisesone or more CrossFabs is referred to herein as “CrossMab.”

In contrast thereto, by a “conventional” Fab molecule is meant a Fabmolecule in its natural format, i.e. comprising a heavy chain composedof the heavy chain variable and constant regions (VH-CH1), and a lightchain composed of the light chain variable and constant regions (VL-CL).

The term “immunoglobulin molecule” refers to a protein having thestructure of a naturally occurring antibody. For example,immunoglobulins of the IgG class are heterotetrameric glycoproteins ofabout 150,000 daltons, composed of two light chains and two heavy chainsthat are disulfide-bonded. From N- to C-terminus, each heavy chain has avariable region (VH), also called a variable heavy domain or a heavychain variable domain, followed by three constant domains (CH1, CH2, andCH3), also called a heavy chain constant region. Similarly, from N- toC-terminus, each light chain has a variable region (VL), also called avariable light domain or a light chain variable domain, followed by aconstant light (CL) domain, also called a light chain constant region.The heavy chain of an immunoglobulin may be assigned to one of fivetypes, called α (IgA), δ (IgD), ε (IgE), γ (IgG), or μ (IgM), some ofwhich may be further divided into subtypes, e.g. γ₁ (IgG₁), γ₂ (IgG₂),γ₃ (IgG₃), γ₄ (IgG₄), α₁ (IgA₁) and α₂ (IgA₂). The light chain of animmunoglobulin may be assigned to one of two types, called kappa (κ) andlambda (λ), based on the amino acid sequence of its constant domain. Animmunoglobulin essentially consists of two Fab molecules and an Fcdomain, linked via the immunoglobulin hinge region.

The term “antibody” herein is used in the broadest sense and encompassesvarious antibody structures, including but not limited to monoclonalantibodies, polyclonal antibodies, and antibody fragments so long asthey exhibit the desired antigen-binding activity.

An “antibody fragment” refers to a molecule other than an intactantibody that comprises a portion of an intact antibody that binds theantigen to which the intact antibody binds. Examples of antibodyfragments include but are not limited to Fv, Fab, Fab′, Fab′-SH,F(ab′)₂, diabodies, linear antibodies, single-chain antibody molecules(e.g. scFv), and single-domain antibodies. For a review of certainantibody fragments, see Hudson et al., Nat Med 9, 129-134 (2003). For areview of scFv fragments, see e.g. Plückthun, in The Pharmacology ofMonoclonal Antibodies, vol. 113, Rosenburg and Moore eds.,Springer-Verlag, New York, pp. 269-315 (1994); see also WO 93/16185; andU.S. Pat. Nos. 5,571,894 and 5,587,458. For discussion of Fab andF(ab′)₂ fragments comprising salvage receptor binding epitope residuesand having increased in vivo half-life, see U.S. Pat. No. 5,869,046.Diabodies are antibody fragments with two antigen-binding sites that maybe bivalent or bispecific. See, for example, EP 404,097; WO 1993/01161;Hudson et al., Nat Med 9, 129-134 (2003); and Hollinger et al., ProcNatl Acad Sci USA 90, 6444-6448 (1993). Triabodies and tetrabodies arealso described in Hudson et al., Nat Med 9, 129-134 (2003).Single-domain antibodies are antibody fragments comprising all or aportion of the heavy chain variable domain or all or a portion of thelight chain variable domain of an antibody. In certain embodiments, asingle-domain antibody is a human single-domain antibody (Domantis,Inc., Waltham, Mass.; see e.g. U.S. Pat. No. 6,248,516 B1). Antibodyfragments can be made by various techniques, including but not limitedto proteolytic digestion of an intact antibody as well as production byrecombinant host cells (e.g. E. coli or phage), as described herein.

The term “antigen binding domain” refers to the part of an antibody thatcomprises the area which specifically binds to and is complementary topart or all of an antigen. An antigen binding domain may be provided by,for example, one or more antibody variable domains (also called antibodyvariable regions). Particularly, an antigen binding domain comprises anantibody light chain variable region (VL) and an antibody heavy chainvariable region (VH).

The term “variable region” or “variable domain” refers to the domain ofan antibody heavy or light chain that is involved in binding theantibody to antigen. The variable domains of the heavy chain and lightchain (VH and VL, respectively) of a native antibody generally havesimilar structures, with each domain comprising four conserved frameworkregions (FRs) and three hypervariable regions (HVRs). See, e.g., Kindtet al., Kuby Immunology, 6^(th) ed., W.H. Freeman and Co., page 91(2007). A single VH or VL domain may be sufficient to conferantigen-binding specificity.

The term “hypervariable region” or “HVR”, as used herein, refers to eachof the regions of an antibody variable domain which are hypervariable insequence and/or form structurally defined loops (“hypervariable loops”).Generally, native four-chain antibodies comprise six HVRs; three in theVH (H1, H2, H3), and three in the VL (L1, L2, L3). HVRs generallycomprise amino acid residues from the hypervariable loops and/or fromthe complementarity determining regions (CDRs), the latter being ofhighest sequence variability and/or involved in antigen recognition.With the exception of CDR1 in VH, CDRs generally comprise the amino acidresidues that form the hypervariable loops. Hypervariable regions (HVRs)are also referred to as “complementarity determining regions” (CDRs),and these terms are used herein interchangeably in reference to portionsof the variable region that form the antigen binding regions. Thisparticular region has been described by Kabat et al., U.S. Dept. ofHealth and Human Services, Sequences of Proteins of ImmunologicalInterest (1983) and by Chothia et al., J Mol Biol 196:901-917 (1987),where the definitions include overlapping or subsets of amino acidresidues when compared against each other. Nevertheless, application ofeither definition to refer to a CDR of an antibody or variants thereofis intended to be within the scope of the term as defined and usedherein. The appropriate amino acid residues which encompass the CDRs asdefined by each of the above cited references are set forth below inTable A as a comparison. The exact residue numbers which encompass aparticular CDR will vary depending on the sequence and size of the CDR.Those skilled in the art can routinely determine which residues comprisea particular CDR given the variable region amino acid sequence of theantibody.

TABLE A CDR Definitions¹ CDR Kabat Chothia AbM² V_(H) CDR1 31-35 26-3226-35 V_(H) CDR2 50-65 52-58 50-58 V_(H) CDR3  95-102  95-102  95-102V_(L) CDR1 24-34 26-32 24-34 V_(L) CDR2 50-56 50-52 50-56 V_(L) CDR389-97 91-96 89-97 ¹Numbering of all CDR definitions in Table A isaccording to the numbering conventions set forth by Kabat et al. (seebelow). ²“AbM” with a lowercase “b” as used in Table A refers to theCDRs as defined by Oxford Molecular's “AbM” antibody modeling software.

Kabat et al. also defined a numbering system for variable regionsequences that is applicable to any antibody. One of ordinary skill inthe art can unambiguously assign this system of “Kabat numbering” to anyvariable region sequence, without reliance on any experimental databeyond the sequence itself. As used herein, “Kabat numbering” refers tothe numbering system set forth by Kabat et al., U.S. Dept. of Health andHuman Services, “Sequence of Proteins of Immunological Interest” (1983).Unless otherwise specified, references to the numbering of specificamino acid residue positions in an antibody variable region areaccording to the Kabat numbering system. The polypeptide sequences ofthe sequence listing are not numbered according to the Kabat numberingsystem. However, it is well within the ordinary skill of one in the artto convert the numbering of the sequences of the Sequence Listing toKabat numbering.

“Framework” or “FR” refers to variable domain residues other thanhypervariable region (HVR) residues. The FR of a variable domaingenerally consists of four FR domains: FR1, FR2, FR3, and FR4.Accordingly, the HVR and FR sequences generally appear in the followingsequence in VH (or VL): FR1-H1(L1)-FR2-H2(L2)-FR3-H3 (L3)-FR4.

The “class” of an antibody or immunoglobulin refers to the type ofconstant domain or constant region possessed by its heavy chain. Thereare five major classes of antibodies: IgA, IgD, IgE, IgG, and IgM, andseveral of these may be further divided into subclasses (isotypes),e.g., IgG₁, IgG₂, IgG₃, IgG₄, IgA₁, and IgA₂. The heavy chain constantdomains that correspond to the different classes of immunoglobulins arecalled α, δ, ε, γ, and μ, respectively.

The term “Fc domain” or “Fc region” herein is used to define aC-terminal region of an immunoglobulin heavy chain that contains atleast a portion of the constant region. The term includes nativesequence Fc regions and variant Fc regions. Although the boundaries ofthe Fc region of an IgG heavy chain might vary slightly, the human IgGheavy chain Fc region is usually defined to extend from Cys226, or fromPro230, to the carboxyl-terminus of the heavy chain. However, theC-terminal lysine (Lys447) of the Fc region may or may not be present.Unless otherwise specified herein, numbering of amino acid residues inthe Fc region or constant region is according to the EU numberingsystem, also called the EU index, as described in Kabat et al.,Sequences of Proteins of Immunological Interest, 5th Ed. Public HealthService, National Institutes of Health, Bethesda, Md., 1991. A “subunit”of an Fc domain as used herein refers to one of the two polypeptidesforming the dimeric Fc domain, i.e. a polypeptide comprising C-terminalconstant regions of an immunoglobulin heavy chain, capable of stableself-association. For example, a subunit of an IgG Fc domain comprisesan IgG CH2 and an IgG CH3 constant domain.

A “modification promoting the association of the first and the secondsubunit of the Fc domain” is a manipulation of the peptide backbone orthe post-translational modifications of an Fc domain subunit thatreduces or prevents the association of a polypeptide comprising the Fcdomain subunit with an identical polypeptide to form a homodimer. Amodification promoting association as used herein particularly includesseparate modifications made to each of the two Fc domain subunitsdesired to associate (i.e. the first and the second subunit of the Fcdomain), wherein the modifications are complementary to each other so asto promote association of the two Fc domain subunits. For example, amodification promoting association may alter the structure or charge ofone or both of the Fc domain subunits so as to make their associationsterically or electrostatically favorable, respectively. Thus,(hetero)dimerization occurs between a polypeptide comprising the firstFc domain subunit and a polypeptide comprising the second Fc domainsubunit, which might be non-identical in the sense that furthercomponents fused to each of the subunits (e.g. antigen binding moieties)are not the same. In some embodiments the modification promotingassociation comprises an amino acid mutation in the Fc domain,specifically an amino acid substitution. In a particular embodiment, themodification promoting association comprises a separate amino acidmutation, specifically an amino acid substitution, in each of the twosubunits of the Fc domain.

The term “effector functions” refers to those biological activitiesattributable to the Fc region of an antibody, which vary with theantibody isotype. Examples of antibody effector functions include: C1qbinding and complement dependent cytotoxicity (CDC), Fc receptorbinding, antibody-dependent cell-mediated cytotoxicity (ADCC),antibody-dependent cellular phagocytosis (ADCP), cytokine secretion,immune complex-mediated antigen uptake by antigen presenting cells, downregulation of cell surface receptors (e.g. B cell receptor), and B cellactivation.

As used herein, the terms “engineer, engineered, engineering”, areconsidered to include any manipulation of the peptide backbone or thepost-translational modifications of a naturally occurring or recombinantpolypeptide or fragment thereof. Engineering includes modifications ofthe amino acid sequence, of the glycosylation pattern, or of the sidechain group of individual amino acids, as well as combinations of theseapproaches.

The term “amino acid mutation” as used herein is meant to encompassamino acid substitutions, deletions, insertions, and modifications. Anycombination of substitution, deletion, insertion, and modification canbe made to arrive at the final construct, provided that the finalconstruct possesses the desired characteristics, e.g., reduced bindingto an Fc receptor, or increased association with another peptide. Aminoacid sequence deletions and insertions include amino- and/orcarboxy-terminal deletions and insertions of amino acids. Particularamino acid mutations are amino acid substitutions. For the purpose ofaltering e.g. the binding characteristics of an Fc region,non-conservative amino acid substitutions, i.e. replacing one amino acidwith another amino acid having different structural and/or chemicalproperties, are particularly preferred. Amino acid substitutions includereplacement by non-naturally occurring amino acids or by naturallyoccurring amino acid derivatives of the twenty standard amino acids(e.g. 4-hydroxyproline, 3-methylhistidine, ornithine, homoserine,5-hydroxylysine). Amino acid mutations can be generated using genetic orchemical methods well known in the art. Genetic methods may includesite-directed mutagenesis, PCR, gene synthesis and the like. It iscontemplated that methods of altering the side chain group of an aminoacid by methods other than genetic engineering, such as chemicalmodification, may also be useful. Various designations may be usedherein to indicate the same amino acid mutation. For example, asubstitution from proline at position 329 of the Fc domain to glycinecan be indicated as 329G, G329, G₃₂₉, P329G, or Pro329Gly.

As used herein, term “polypeptide” refers to a molecule composed ofmonomers (amino acids) linearly linked by amide bonds (also known aspeptide bonds). The term “polypeptide” refers to any chain of two ormore amino acids, and does not refer to a specific length of theproduct. Thus, peptides, dipeptides, tripeptides, oligopeptides,“protein,” “amino acid chain,” or any other term used to refer to achain of two or more amino acids, are included within the definition of“polypeptide,” and the term “polypeptide” may be used instead of, orinterchangeably with any of these terms. The term “polypeptide” is alsointended to refer to the products of post-expression modifications ofthe polypeptide, including without limitation glycosylation,acetylation, phosphorylation, amidation, derivatization by knownprotecting/blocking groups, proteolytic cleavage, or modification bynon-naturally occurring amino acids. A polypeptide may be derived from anatural biological source or produced by recombinant technology, but isnot necessarily translated from a designated nucleic acid sequence. Itmay be generated in any manner, including by chemical synthesis. Apolypeptide of the invention may be of a size of about 3 or more, 5 ormore, 10 or more, 20 or more, 25 or more, 50 or more, 75 or more, 100 ormore, 200 or more, 500 or more, 1,000 or more, or 2,000 or more aminoacids. Polypeptides may have a defined three-dimensional structure,although they do not necessarily have such structure. Polypeptides witha defined three-dimensional structure are referred to as folded, andpolypeptides which do not possess a defined three-dimensional structure,but rather can adopt a large number of different conformations, and arereferred to as unfolded.

By an “isolated” polypeptide or a variant, or derivative thereof isintended a polypeptide that is not in its natural milieu. No particularlevel of purification is required. For example, an isolated polypeptidecan be removed from its native or natural environment. Recombinantlyproduced polypeptides and proteins expressed in host cells areconsidered isolated for the purpose of the invention, as are native orrecombinant polypeptides which have been separated, fractionated, orpartially or substantially purified by any suitable technique.

“Percent (%) amino acid sequence identity” with respect to a referencepolypeptide sequence is defined as the percentage of amino acid residuesin a candidate sequence that are identical with the amino acid residuesin the reference polypeptide sequence, after aligning the sequences andintroducing gaps, if necessary, to achieve the maximum percent sequenceidentity, and not considering any conservative substitutions as part ofthe sequence identity. Alignment for purposes of determining percentamino acid sequence identity can be achieved in various ways that arewithin the skill in the art, for instance, using publicly availablecomputer software such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR)software. Those skilled in the art can determine appropriate parametersfor aligning sequences, including any algorithms needed to achievemaximal alignment over the full length of the sequences being compared.For purposes herein, however, % amino acid sequence identity values aregenerated using the sequence comparison computer program ALIGN-2. TheALIGN-2 sequence comparison computer program was authored by Genentech,Inc., and the source code has been filed with user documentation in theU.S. Copyright Office, Washington D.C., 20559, where it is registeredunder U.S. Copyright Registration No. TXU510087. The ALIGN-2 program ispublicly available from Genentech, Inc., South San Francisco, Calif., ormay be compiled from the source code. The ALIGN-2 program should becompiled for use on a UNIX operating system, including digital UNIXV4.0D. All sequence comparison parameters are set by the ALIGN-2 programand do not vary. In situations where ALIGN-2 is employed for amino acidsequence comparisons, the % amino acid sequence identity of a givenamino acid sequence A to, with, or against a given amino acid sequence B(which can alternatively be phrased as a given amino acid sequence Athat has or comprises a certain % amino acid sequence identity to, with,or against a given amino acid sequence B) is calculated as follows:

100 times the fraction X/Y

where X is the number of amino acid residues scored as identical matchesby the sequence alignment program ALIGN-2 in that program's alignment ofA and B, and where Y is the total number of amino acid residues in B. Itwill be appreciated that where the length of amino acid sequence A isnot equal to the length of amino acid sequence B, the % amino acidsequence identity of A to B will not equal the % amino acid sequenceidentity of B to A. Unless specifically stated otherwise, all % aminoacid sequence identity values used herein are obtained as described inthe immediately preceding paragraph using the ALIGN-2 computer program.

The term “polynucleotide” refers to an isolated nucleic acid molecule orconstruct, e.g. messenger RNA (mRNA), virally-derived RNA, or plasmidDNA (pDNA). A polynucleotide may comprise a conventional phosphodiesterbond or a non-conventional bond (e.g. an amide bond, such as found inpeptide nucleic acids (PNA). The term “nucleic acid molecule” refers toany one or more nucleic acid segments, e.g. DNA or RNA fragments,present in a polynucleotide.

By “isolated” nucleic acid molecule or polynucleotide is intended anucleic acid molecule, DNA or RNA, which has been removed from itsnative environment. For example, a recombinant polynucleotide encoding apolypeptide contained in a vector is considered isolated for thepurposes of the present invention. Further examples of an isolatedpolynucleotide include recombinant polynucleotides maintained inheterologous host cells or purified (partially or substantially)polynucleotides in solution. An isolated polynucleotide includes apolynucleotide molecule contained in cells that ordinarily contain thepolynucleotide molecule, but the polynucleotide molecule is presentextrachromosomally or at a chromosomal location that is different fromits natural chromosomal location. Isolated RNA molecules include in vivoor in vitro RNA transcripts of the present invention, as well aspositive and negative strand forms, and double-stranded forms. Isolatedpolynucleotides or nucleic acids according to the present inventionfurther include such molecules produced synthetically. In addition, apolynucleotide or a nucleic acid may be or may include a regulatoryelement such as a promoter, ribosome binding site, or a transcriptionterminator. By a nucleic acid or polynucleotide having a nucleotidesequence at least, for example, 95% “identical” to a referencenucleotide sequence of the present invention, it is intended that thenucleotide sequence of the polynucleotide is identical to the referencesequence except that the polynucleotide sequence may include up to fivepoint mutations per each 100 nucleotides of the reference nucleotidesequence. In other words, to obtain a polynucleotide having a nucleotidesequence at least 95% identical to a reference nucleotide sequence, upto 5% of the nucleotides in the reference sequence may be deleted orsubstituted with another nucleotide, or a number of nucleotides up to 5%of the total nucleotides in the reference sequence may be inserted intothe reference sequence. These alterations of the reference sequence mayoccur at the 5′ or 3′ terminal positions of the reference nucleotidesequence or anywhere between those terminal positions, interspersedeither individually among residues in the reference sequence or in oneor more contiguous groups within the reference sequence. As a practicalmatter, whether any particular polynucleotide sequence is at least 80%,85%, 90%, 95%, 96%, 97%, 98% or 99% identical to a nucleotide sequenceof the present invention can be determined conventionally using knowncomputer programs, such as the ones discussed above for polypeptides(e.g. ALIGN-2).

The term “expression cassette” refers to a polynucleotide generatedrecombinantly or synthetically, with a series of specified nucleic acidelements that permit transcription of a particular nucleic acid in atarget cell. The recombinant expression cassette can be incorporatedinto a plasmid, chromosome, mitochondrial DNA, plastid DNA, virus, ornucleic acid fragment. Typically, the recombinant expression cassetteportion of an expression vector includes, among other sequences, anucleic acid sequence to be transcribed and a promoter. In certainembodiments, the expression cassette of the invention comprisespolynucleotide sequences that encode bispecific antigen bindingmolecules of the invention or fragments thereof.

The term “vector” or “expression vector” is synonymous with “expressionconstruct” and refers to a DNA molecule that is used to introduce anddirect the expression of a specific gene to which it is operablyassociated in a target cell. The term includes the vector as aself-replicating nucleic acid structure as well as the vectorincorporated into the genome of a host cell into which it has beenintroduced. The expression vector of the present invention comprises anexpression cassette. Expression vectors allow transcription of largeamounts of stable mRNA. Once the expression vector is inside the targetcell, the ribonucleic acid molecule or protein that is encoded by thegene is produced by the cellular transcription and/or translationmachinery. In one embodiment, the expression vector of the inventioncomprises an expression cassette that comprises polynucleotide sequencesthat encode bispecific antigen binding molecules of the invention orfragments thereof.

The terms “host cell”, “host cell line,” and “host cell culture” areused interchangeably and refer to cells into which exogenous nucleicacid has been introduced, including the progeny of such cells. Hostcells include “transformants” and “transformed cells,” which include theprimary transformed cell and progeny derived therefrom without regard tothe number of passages. Progeny may not be completely identical innucleic acid content to a parent cell, but may contain mutations. Mutantprogeny that have the same function or biological activity as screenedor selected for in the originally transformed cell are included herein.A host cell is any type of cellular system that can be used to generatethe bispecific antigen binding molecules of the present invention. Hostcells include cultured cells, e.g. mammalian cultured cells, such as CHOcells, BHK cells, NS0 cells, SP2/0 cells, YO myeloma cells, P3X63 mousemyeloma cells, PER cells, PER.C6 cells or hybridoma cells, yeast cells,insect cells, and plant cells, to name only a few, but also cellscomprised within a transgenic animal, transgenic plant or cultured plantor animal tissue.

An “activating Fc receptor” is an Fc receptor that following engagementby an Fc domain of an antibody elicits signaling events that stimulatethe receptor-bearing cell to perform effector functions. Humanactivating Fc receptors include FcγRIIIa (CD16a), FcγRI (CD64), FcγRIIa(CD32), and FcαRI (CD89).

Antibody-dependent cell-mediated cytotoxicity (ADCC) is an immunemechanism leading to the lysis of antibody-coated target cells by immuneeffector cells. The target cells are cells to which antibodies orderivatives thereof comprising an Fc region specifically bind, generallyvia the protein part that is N-terminal to the Fc region. As usedherein, the term “reduced ADCC” is defined as either a reduction in thenumber of target cells that are lysed in a given time, at a givenconcentration of antibody in the medium surrounding the target cells, bythe mechanism of ADCC defined above, and/or an increase in theconcentration of antibody in the medium surrounding the target cells,required to achieve the lysis of a given number of target cells in agiven time, by the mechanism of ADCC. The reduction in ADCC is relativeto the ADCC mediated by the same antibody produced by the same type ofhost cells, using the same standard production, purification,formulation and storage methods (which are known to those skilled in theart), but that has not been engineered. For example the reduction inADCC mediated by an antibody comprising in its Fc domain an amino acidsubstitution that reduces ADCC, is relative to the ADCC mediated by thesame antibody without this amino acid substitution in the Fc domain.Suitable assays to measure ADCC are well known in the art (see e.g. PCTpublication no. WO 2006/082515 or PCT publication no. WO 2012/130831).

An “effective amount” of an agent refers to the amount that is necessaryto result in a physiological change in the cell or tissue to which it isadministered.

A “therapeutically effective amount” of an agent, e.g. a pharmaceuticalcomposition, refers to an amount effective, at dosages and for periodsof time necessary, to achieve the desired therapeutic or prophylacticresult. A therapeutically effective amount of an agent for exampleeliminates, decreases, delays, minimizes or prevents adverse effects ofa disease.

An “individual” or “subject” is a mammal. Mammals include, but are notlimited to, domesticated animals (e.g. cows, sheep, cats, dogs, andhorses), primates (e.g. humans and non-human primates such as monkeys),rabbits, and rodents (e.g. mice and rats). Particularly, the individualor subject is a human.

The term “pharmaceutical composition” refers to a preparation which isin such form as to permit the biological activity of an activeingredient contained therein to be effective, and which contains noadditional components which are unacceptably toxic to a subject to whichthe formulation would be administered.

A “pharmaceutically acceptable carrier” refers to an ingredient in apharmaceutical composition, other than an active ingredient, which isnontoxic to a subject. A pharmaceutically acceptable carrier includes,but is not limited to, a buffer, excipient, stabilizer, or preservative.

As used herein, “treatment” (and grammatical variations thereof such as“treat” or “treating”) refers to clinical intervention in an attempt toalter the natural course of a disease in the individual being treated,and can be performed either for prophylaxis or during the course ofclinical pathology. Desirable effects of treatment include, but are notlimited to, preventing occurrence or recurrence of disease, alleviationof symptoms, diminishment of any direct or indirect pathologicalconsequences of the disease, preventing metastasis, decreasing the rateof disease progression, amelioration or palliation of the disease state,and remission or improved prognosis. In some embodiments, T cellactivating bispecific antigen binding molecules of the invention areused to delay development of a disease or to slow the progression of adisease.

The term “antagonist” is used in the broadest sense, and includes anymolecule that partially or fully blocks, inhibits, or neutralizes abiological activity of a native polypeptide disclosed herein. In asimilar manner, the term “agonist” is used in the broadest sense andincludes any molecule that induces a biological activity of a nativepolypeptide disclosed herein. Suitable agonist or antagonist moleculesspecifically include agonist or antagonist antibodies or antibodyfragments, including engineered antibody fragments, fragments or aminoacid sequence variants of native polypeptides, peptides, antisenseoligonucleotides, small organic molecules, etc. Methods for identifyingagonists or antagonists of a polypeptide may comprise contacting apolypeptide with a candidate agonist or antagonist molecule andmeasuring a detectable change in one or more biological activitiesnormally associated with the polypeptide.

The term “package insert” is used to refer to instructions customarilyincluded in commercial packages of therapeutic products, that containinformation about the indications, usage, dosage, administration,combination therapy, contraindications and/or warnings concerning theuse of such therapeutic products.

All references, publication, patents and patent applications disclosedherein are hereby incorporated by reference in their entirety.

DETAILED DESCRIPTION OF THE EMBODIMENTS

The T cell activating bispecific antigen binding molecule of theinvention is bispecific, i.e. it comprises at least two antigen bindingmoieties capable of specific binding to two distinct antigenicdeterminants, i.e. to CD3 and to FolR1. According to the invention, theantigen binding moieties are Fab molecules (i.e. antigen binding domainscomposed of a heavy and a light chain, each comprising a variable and aconstant region). In one embodiment said Fab molecules are human. Inanother embodiment said Fab molecules are humanized. In yet anotherembodiment said Fab molecules comprise human heavy and light chainconstant regions.

The T cell activating bispecific antigen binding molecule of theinvention is capable of simultaneous binding to the target cell antigenFolR1 and CD3. In one embodiment, the T cell activating bispecificantigen binding molecule is capable of crosslinking a T cell and a FolR1expressing target cell by simultaneous binding to the target cellantigen FolR1 and CD3. In an even more particular embodiment, suchsimultaneous binding results in lysis of the FolR1 expressing targetcell, particularly a FolR1 expressing tumor cell. In one embodiment,such simultaneous binding results in activation of the T cell. In otherembodiments, such simultaneous binding results in a cellular response ofa T lymphocyte, particularly a cytotoxic T lymphocyte, selected from thegroup of: proliferation, differentiation, cytokine secretion, cytotoxiceffector molecule release, cytotoxic activity, and expression ofactivation markers. In one embodiment, binding of the T cell activatingbispecific antigen binding molecule to CD3 without simultaneous bindingto the target cell antigen FolR1 does not result in T cell activation.

In one embodiment, the T cell activating bispecific antigen bindingmolecule is capable of re-directing cytotoxic activity of a T cell to aFolR1 expressing target cell. In a particular embodiment, saidre-direction is independent of MHC-mediated peptide antigen presentationby the target cell and and/or specificity of the T cell.

Particularly, a T cell according to some of the embodiments of theinvention is a cytotoxic T cell. In some embodiments the T cell is aCD4⁺ or a CD8⁺ T cell, particularly a CD8⁺ T cell.

The T cell activating bispecific antigen binding molecule of theinvention comprises at least one antigen binding moiety capable ofbinding to CD3 (also referred to herein as an “CD3 antigen bindingmoiety” or “first antigen binding moiety”). In a particular embodiment,the T cell activating bispecific antigen binding molecule comprises notmore than one antigen binding moiety capable of specific binding to CD3.In one embodiment the T cell activating bispecific antigen bindingmolecule provides monovalent binding to CD3. In a particular embodimentCD3 is human CD3 or cynomolgus CD3, most particularly human CD3. In aparticular embodiment the CD3 antigen binding moiety is cross-reactivefor (i.e. specifically binds to) human and cynomolgus CD3. In someembodiments, the first antigen binding moiety is capable of specificbinding to the epsilon subunit of CD3 (see UniProt no. P07766 (version130), NCBI RefSeq no. NP_000724.1, SEQ ID NO:150 for the human sequence;UniProt no. Q95LI5 (version 49), NCBI GenBank no. BAB71849.1, for thecynomolgus [Macaca fascicularis] sequence).

In some embodiments, the CD3 antigen binding moiety comprises at leastone heavy chain complementarity determining region (CDR) selected fromthe group consisting of SEQ ID NO: 37, SEQ ID NO: 38 and SEQ ID NO: 39and at least one light chain CDR selected from the group of SEQ ID NO:32, SEQ ID NO: 33, SEQ ID NO: 34.

In one embodiment the CD3 antigen binding moiety comprises the heavychain CDR1 of SEQ ID NO: 37, the heavy chain CDR2 of SEQ ID NO: 38, theheavy chain CDR3 of SEQ ID NO:39, the light chain CDR1 of SEQ ID NO: 32,the light chain CDR2 of SEQ ID NO: 33, and the light chain CDR3 of SEQID NO:34.

In one embodiment the CD3 antigen binding moiety comprises a variableheavy chain comprising an amino acid sequence of: SEQ ID NO: 36 and avariable light chain comprising an amino acid sequence of: SEQ ID NO:31.

In one embodiment the CD3 antigen binding moiety comprises a heavy chainvariable region sequence that is at least about 95%, 96%, 97%, 98%, 99%or 100% identical to SEQ ID NO: 36 and a light chain variable regionsequence that is at least about 95%, 96%, 97%, 98%, 99% or 100%identical to SEQ ID NO: 31.

The T cell activating bispecific antigen binding molecule of theinvention comprises at least one antigen binding moiety capable ofbinding to the target cell antigen FolR1 (also referred to herein as an“FolR1 binding moiety” or “second” or “third” antigen binding moiety).In one embodiment, the antigen binding moiety capable of binding to thetarget cell antigen FolR1 does not bind to FolR2 or FolR3. In aparticular embodiment the FolR1 antigen binding moiety is cross-reactivefor (i.e. specifically binds to) human and cynomolgus FolR1. In certainembodiments, the T cell activating bispecific antigen binding moleculecomprises two antigen binding moieties capable of binding to the targetcell antigen FolR1. In a particular such embodiment, each of theseantigen binding moieties specifically binds to the same antigenicdeterminant. In an even more particular embodiment, all of these antigenbinding moieties are identical. In one embodiment the T cell activatingbispecific antigen binding molecule comprises not more than two antigenbinding moieties capable of binding to FolR1.

The FolR1 binding moiety is generally a Fab molecule that specificallybinds to FolR1 and is able to direct the T cell activating bispecificantigen binding molecule to which it is connected to a target site, forexample to a specific type of tumor cell that expresses FolR1.

In one aspect the present invention provides a T cell activatingbispecific antigen binding molecule comprising

-   -   (i) a first antigen binding moiety which is a Fab molecule        capable of specific binding to CD3, and which comprises at least        one heavy chain complementarity determining region (CDR)        selected from the group consisting SEQ ID NO: 37, SEQ ID NO: 38        and SEQ ID NO: 39 and at least one light chain CDR selected from        the group of SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34; and    -   (ii) a second antigen binding moiety which is a Fab molecule        capable of specific binding to Folate Receptor 1 (FolR1).

In one embodiment the first antigen binding moiety which is a Fabmolecule capable of specific binding to CD3 comprises a variable heavychain comprising an amino acid sequence of SEQ ID NO: 36 and a variablelight chain comprising an amino acid sequence of SEQ ID NO: 31.

In one embodiment the T cell activating bispecific antigen bindingmolecule additionally comprises

-   -   (iii) a third antigen binding moiety which is a Fab molecule        capable of specific binding to FolR1.

In one such embodiment the second and third antigen binding moietycapable of specific binding to FolR1 comprise identical heavy chaincomplementarity determining region (CDR) and light chain CDR sequences.In one such embodiment the third antigen binding moiety is identical tothe second antigen binding moiety.

In one embodiment the T cell activating bispecific antigen bindingmolecule of any of the above embodiments additionally comprises an Fcdomain composed of a first and a second subunit capable of stableassociation.

In one embodiment the first antigen binding moiety and the secondantigen binding moiety are each fused at the C-terminus of the Fab heavychain to the N-terminus of the first or second subunit of the Fc domain.

In one embodiment the third antigen binding moiety is fused at theC-terminus of the Fab heavy chain to the N-terminus of the Fab heavychain of the first antigen binding moiety, optionally via a peptidelinker.

In a further particular embodiment, not more than one antigen bindingmoiety capable of specific binding to CD3 is present in the T cellactivating bispecific antigen binding molecule (i.e. the T cellactivating bispecific antigen binding molecule provides monovalentbinding to CD3).

T Cell Activating Bispecific Antigen Binding Molecule with a CommonLight Chain

The inventors of the present invention generated a bispecific antibodywherein the binding moieties share a common light chain that retains thespecificity and efficacy of the parent monospecific antibody for CD3 andcan bind a second antigen (e.g., FolR1) using the same light chain. Thegeneration of a bispecific molecule with a common light chain thatretains the binding properties of the parent antibody is notstraight-forward as the common CDRs of the hybrid light chain have toeffectuate the binding specificity for both targets. In one aspect thepresent invention provides a T cell activating bispecific antigenbinding molecule comprising a first and a second antigen binding moiety,one of which is a Fab molecule capable of specific binding to CD3 andthe other one of which is a Fab molecule capable of specific binding toFolR1, wherein the first and the second Fab molecule have identical VLCLlight chains. In one embodiment said identical light chain (VLCL)comprises the light chain CDRs of SEQ ID NO: 32, SEQ ID NO: 33 and SEQID NO: 34. In one embodiment said identical light chain (VLCL) comprisesSEQ ID NO. 35.

In one embodiment the present invention provides a T cell activatingbispecific antigen binding molecule comprising

(i) a first antigen binding moiety which is a Fab molecule capable ofspecific binding to CD3, and which comprises at least one heavy chaincomplementarity determining region (CDR) selected from the groupconsisting of SEQ ID NO: 37, SEQ ID NO: 38 and SEQ ID NO: 39 and atleast one light chain CDR selected from the group of SEQ ID NO: 32, SEQID NO: 33, SEQ ID NO: 34;

(ii) a second antigen binding moiety which is a Fab molecule capable ofspecific binding to Folate Receptor 1 (FolR1) and which comprises atleast one heavy chain complementarity determining region (CDR) selectedfrom the group consisting of SEQ ID NO: 16, SEQ ID NO: 17 and SEQ ID NO:18 and at least one light chain CDR selected from the group of SEQ IDNO: 32, SEQ ID NO: 33, SEQ ID NO: 34.

In one such embodiment the CD3 antigen binding moiety comprises theheavy chain CDR1 of SEQ ID NO: 37, the heavy chain CDR2 of SEQ ID NO:38, the heavy chain CDR3 of SEQ ID NO:39, the light chain CDR1 of SEQ IDNO: 32, the light chain CDR2 of SEQ ID NO: 33, and the light chain CDR3of SEQ ID NO:34 and the FolR1 antigen binding moiety comprises the heavychain CDR1 of SEQ ID NO: 16, the heavy chain CDR2 of SEQ ID NO: 17, theheavy chain CDR3 of SEQ ID NO:18, the light chain CDR1 of SEQ ID NO: 32,the light chain CDR2 of SEQ ID NO: 33, and the light chain CDR3 of SEQID NO:34.

In one embodiment the present invention provides a T cell activatingbispecific antigen binding molecule comprising

(i) a first antigen binding moiety which is a Fab molecule capable ofspecific binding to CD3 comprising a variable heavy chain comprising anamino acid sequence of SEQ ID NO: 36 and a variable light chaincomprising an amino acid sequence of SEQ ID NO: 31.

(ii) a second antigen binding moiety which is a Fab molecule capable ofspecific binding to Folate Receptor 1 (FolR1) comprising a variableheavy chain comprising an amino acid sequence of SEQ ID NO: 15 and avariable light chain comprising an amino acid sequence of SEQ ID NO: 31.

In one embodiment the present invention provides a T cell activatingbispecific antigen binding molecule comprising

(i) a first antigen binding moiety which is a Fab molecule capable ofspecific binding to CD3, and which comprises at least one heavy chaincomplementarity determining region (CDR) selected from the groupconsisting of SEQ ID NO: 37, SEQ ID NO: 38 and SEQ ID NO: 39 and atleast one light chain CDR selected from the group of SEQ ID NO: 32, SEQID NO: 33, SEQ ID NO: 34;

(ii) a second antigen binding moiety which is a Fab molecule capable ofspecific binding to Folate Receptor 1 (FolR1) and which comprises atleast one heavy chain complementarity determining region (CDR) selectedfrom the group consisting of SEQ ID NO: 16, SEQ ID NO: 275 and SEQ IDNO: 315 and at least one light chain CDR selected from the group of SEQID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34.

In one embodiment the present invention provides a T cell activatingbispecific antigen binding molecule comprising

(i) a first antigen binding moiety which is a Fab molecule capable ofspecific binding to CD3, and which comprises the heavy chaincomplementarity determining region (CDR) amino acid sequences of SEQ IDNO: 37, SEQ ID NO: 38 and SEQ ID NO: 39, and the light chain CDR aminoacid sequences of SEQ ID NO: 32, SEQ ID NO: 33 and SEQ ID NO: 34;

(ii) a second antigen binding moiety which is a Fab molecule capable ofspecific binding to Folate Receptor 1 (FolR1) and which comprises theheavy chain complementarity determining region (CDR) amino acidsequences of SEQ ID NO: 16, SEQ ID NO: 275 and SEQ ID NO: 315, and thelight chain CDR amino acid sequences of SEQ ID NO: 32, SEQ ID NO: 33,and SEQ ID NO: 34.

In one embodiment the present invention provides a T cell activatingbispecific antigen binding molecule comprising

(i) a first antigen binding moiety which is a Fab molecule capable ofspecific binding to CD3 comprising a variable heavy chain comprising anamino acid sequence of SEQ ID NO: 36 and a variable light chaincomprising an amino acid sequence of SEQ ID NO: 31;

(ii) a second antigen binding moiety which is a Fab molecule capable ofspecific binding to Folate Receptor 1 (FolR1) comprising a variableheavy chain comprising an amino acid sequence of SEQ ID NO: 274 and avariable light chain comprising an amino acid sequence of SEQ ID NO: 31.

In one embodiment the present invention provides a T cell activatingbispecific antigen binding molecule comprising

(i) a first antigen binding moiety which is a Fab molecule capable ofspecific binding to CD3 comprising a variable heavy chain comprising anamino acid sequence of SEQ ID NO: 36 and a variable light chaincomprising an amino acid sequence of SEQ ID NO: 31.

(ii) a second antigen binding moiety which is a Fab molecule capable ofspecific binding to Folate Receptor 1 (FolR1) comprising a variableheavy chain comprising an amino acid sequence of SEQ ID NO: 15 and avariable light chain comprising an amino acid sequence of SEQ ID NO: 31.

In a further embodiment, the antigen binding moiety that is specific forFolR1 comprises a heavy chain variable region sequence that is at leastabout 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO:15 and alight chain variable region sequence that is at least about 95%, 96%,97%, 98%, 99% or 100% identical to SEQ ID NO: 31 or variants thereofthat retain functionality.

In one embodiment the T cell activating bispecific antigen bindingmolecule comprises a polypeptide sequence that is at least about 95%,96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 36, a polypeptidesequence that is at least about 95%, 96%, 97%, 98%, 99% or 100%identical to SEQ ID NO:15, and a polypeptide sequence that is at leastabout 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 31.

In one embodiment the T cell activating bispecific antigen bindingmolecule additionally comprises

-   -   (iii) a third antigen binding moiety (which is a Fab molecule)        capable of specific binding to FolR1.

In one such embodiment the second and third antigen binding moietycapable of specific binding to FolR1 comprise identical heavy chaincomplementarity determining region (CDR) and light chain CDR sequences.In one such embodiment the third antigen binding moiety is identical tothe second antigen binding moiety.

Hence in one embodiment the present invention provides a T cellactivating bispecific antigen binding molecule comprising

(i) a first antigen binding moiety which is a Fab molecule capable ofspecific binding to CD3, and which comprises at least one heavy chaincomplementarity determining region (CDR) selected from the groupconsisting of SEQ ID NO: 37, SEQ ID NO: 38 and SEQ ID NO: 39 and atleast one light chain CDR selected from the group of SEQ ID NO: 32, SEQID NO: 33, SEQ ID NO: 34;

(ii) a second antigen binding moiety which is a Fab molecule capable ofspecific binding to Folate Receptor 1 (FolR1) and which comprises atleast one heavy chain complementarity determining region (CDR) selectedfrom the group consisting of SEQ ID NO: 16, SEQ ID NO: 17 and SEQ ID NO:18 and at least one light chain CDR selected from the group of SEQ IDNO: 32, SEQ ID NO: 33, SEQ ID NO: 34.

(iii) a third antigen binding moiety which is a Fab molecule capable ofspecific binding to Folate Receptor 1 (FolR1) and which comprises atleast one heavy chain complementarity determining region (CDR) selectedfrom the group consisting of SEQ ID NO: 16, SEQ ID NO: 17 and SEQ ID NO:18 and at least one light chain CDR selected from the group of SEQ IDNO: 32, SEQ ID NO: 33, SEQ ID NO: 34.

In one such embodiment the CD3 antigen binding moiety comprises theheavy chain CDR1 of SEQ ID NO: 37, the heavy chain CDR2 of SEQ ID NO:38, the heavy chain CDR3 of SEQ ID NO:39, the light chain CDR1 of SEQ IDNO: 32, the light chain CDR2 of SEQ ID NO: 33, and the light chain CDR3of SEQ ID NO:34 and the FolR1 antigen binding moiety comprises the heavychain CDR1 of SEQ ID NO: 16, the heavy chain CDR2 of SEQ ID NO: 17, theheavy chain CDR3 of SEQ ID NO:18, the light chain CDR1 of SEQ ID NO: 32,the light chain CDR2 of SEQ ID NO: 33, and the light chain CDR3 of SEQID NO:34.

In one embodiment the present invention provides a T cell activatingbispecific antigen binding molecule comprising

(i) a first antigen binding moiety which is a Fab molecule capable ofspecific binding to CD3 comprising a variable heavy chain comprising anamino acid sequence of SEQ ID NO: 36 and a variable light chaincomprising an amino acid sequence of SEQ ID NO: 31.

(ii) a second antigen binding moiety which is a Fab molecule capable ofspecific binding to Folate Receptor 1 (FolR1) comprising a variableheavy chain comprising an amino acid sequence of SEQ ID NO: 15 and avariable light chain comprising an amino acid sequence of SEQ ID NO: 31.

(iii) a third antigen binding moiety which is a Fab molecule capable ofspecific binding to Folate Receptor 1 (FolR1) comprising a variableheavy chain comprising an amino acid sequence of SEQ ID NO: 15 and avariable light chain comprising an amino acid sequence of SEQ ID NO: 31.

Thus, in one embodiment, the invention relates to bispecific moleculeswherein at least two binding moieties have identical light chains andcorresponding remodeled heavy chains that confer the specific binding tothe T cell activating antigen CD3 and the target cell antigen FolR1,respectively. The use of this so-called ‘common light chain’ principle,i.e. combining two binders that share one light chain but still haveseparate specificities, prevents light chain mispairing. Thus, there areless side products during production, facilitating the homogenouspreparation of T cell activating bispecific antigen binding molecules.

The components of the T cell activating bispecific antigen bindingmolecule can be fused to each other in a variety of configurations.Exemplary configurations are depicted in FIGS. 1A-I and are furtherdescribed below.

In some embodiments, said T cell activating bispecific antigen bindingmolecule further comprises an Fc domain composed of a first and a secondsubunit capable of stable association. Below exemplary embodiments of Tcell activating bispecific antigen binding molecule comprising an Fcdomain are described.

T Cell Activating Bispecific Antigen Binding Molecule with a CrossoverFab Fragment

The inventors of the present invention generated a second bispecificantibody format wherein one of the binding moieties is a crossover Fabfragment. In one aspect of the invention a monovalent bispecificantibody is provided, wherein one of the Fab fragments of an IgGmolecule is replaced by a crossover Fab fragment. Crossover Fabfragments are Fab fragments wherein either the variable regions or theconstant regions of the heavy and light chain are exchanged. Bispecificantibody formats comprising crossover Fab fragments have been described,for example, in WO2009080252, WO2009080253, WO2009080251, WO2009080254,WO2010/136172, WO2010/145792 and WO2013/026831. In a particularembodiment, the first antigen binding moiety is a crossover Fab moleculewherein either the variable or the constant regions of the Fab lightchain and the Fab heavy chain are exchanged. Such modification preventmispairing of heavy and light chains from different Fab molecules,thereby improving the yield and purity of the T cell activatingbispecific antigen binding molecule of the invention in recombinantproduction. In a particular crossover Fab molecule useful for the T cellactivating bispecific antigen binding molecule of the invention, thevariable regions of the Fab light chain and the Fab heavy chain areexchanged. In another crossover Fab molecule useful for the T cellactivating bispecific antigen binding molecule of the invention, theconstant regions of the Fab light chain and the Fab heavy chain areexchanged.

In one embodiment the T cell activating bispecific antigen bindingmolecule comprises

(i) a first antigen binding moiety which is a crossover Fab moleculecapable of specific binding to CD3, comprising at least one heavy chaincomplementarity determining region (CDR) selected from the groupconsisting of SEQ ID NO: 37, SEQ ID NO: 38 and SEQ ID NO: 39 and atleast one light chain CDR selected from the group of SEQ ID NO: 32, SEQID NO: 33, SEQ ID NO: 34;

(ii) a second antigen binding moiety capable of specific binding toFolate Receptor 1 (FolR1) comprising at least one heavy chaincomplementarity determining region (CDR) selected from the groupconsisting of SEQ ID NO: 8, SEQ ID NO: 56 and SEQ ID NO: 57 and at leastone light chain CDR selected from the group of SEQ ID NO: 59, SEQ ID NO:60, SEQ ID NO: 65.

In one such embodiment the CD3 antigen binding moiety comprises theheavy chain CDR1 of SEQ ID NO: 37, the heavy chain CDR2 of SEQ ID NO:38, the heavy chain CDR3 of SEQ ID NO:39, the light chain CDR1 of SEQ IDNO: 32, the light chain CDR2 of SEQ ID NO: 33, and the light chain CDR3of SEQ ID NO:34 and the FolR1 antigen binding moiety comprises the heavychain CDR1 of SEQ ID NO: 8, the heavy chain CDR2 of SEQ ID NO: 56, theheavy chain CDR3 of SEQ ID NO:57, the light chain CDR1 of SEQ ID NO: 59,the light chain CDR2 of SEQ ID NO: 60, and the light chain CDR3 of SEQID NO:65.

In one embodiment, the second antigen binding moiety is a conventionalFab molecule.

In one embodiment the T cell activating bispecific antigen bindingmolecule comprises

(i) a first antigen binding moiety which is a crossover Fab moleculecapable of specific binding to CD3 comprising a variable heavy chaincomprising an amino acid sequence of SEQ ID NO: 36 and a variable lightchain comprising an amino acid sequence of SEQ ID NO: 31.

(ii) a second antigen binding moiety which is a Fab molecule capable ofspecific binding to Folate Receptor 1 (FolR1) comprising a variableheavy chain comprising an amino acid sequence of SEQ ID NO: 55 and avariable light chain comprising an amino acid sequence of SEQ ID NO: 64.

In one embodiment, the second antigen binding moiety is a conventionalFab molecule.

In a further embodiment, the antigen binding moiety that is specific forFolR1 comprises a heavy chain variable region sequence that is at leastabout 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO:55 and alight chain variable region sequence that is at least about 95%, 96%,97%, 98%, 99% or 100% identical to SEQ ID NO: 64 or variants thereofthat retain functionality.

In one embodiment the T cell activating bispecific antigen bindingmolecule comprises a polypeptide sequence that is at least about 95%,96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 36, a polypeptidesequence that is at least about 95%, 96%, 97%, 98%, 99% or 100%identical to SEQ ID NO: 31, a polypeptide sequence that is at leastabout 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO:55, and apolypeptide sequence that is at least about 95%, 96%, 97%, 98%, 99% or100% identical to SEQ ID NO: 64.

In one embodiment the T cell activating bispecific antigen bindingmolecule additionally comprises

-   -   (iii) a third antigen binding moiety capable of specific binding        to FolR1.

In one embodiment, the third antigen binding moiety is a conventionalFab molecule. In one embodiment, the third antigen binding moiety is acrossover Fab molecule.

In one such embodiment the second and third antigen binding moietycapable of specific binding to FolR1 comprise identical heavy chaincomplementarity determining region (CDR) and light chain CDR sequences.In one such embodiment the third antigen binding moiety is identical tothe second antigen binding moiety.

In one embodiment the T cell activating bispecific antigen bindingmolecule comprises

(i) a first antigen binding moiety which is a crossover Fab moleculecapable of specific binding to CD3, comprising at least one heavy chaincomplementarity determining region (CDR) selected from the groupconsisting of SEQ ID NO: 37, SEQ ID NO: 38 and SEQ ID NO: 39 and atleast one light chain CDR selected from the group of SEQ ID NO: 32, SEQID NO: 33, SEQ ID NO: 34;

(ii) a second antigen binding moiety capable of specific binding toFolate Receptor 1 (FolR1) comprising at least one heavy chaincomplementarity determining region (CDR) selected from the groupconsisting of SEQ ID NO: 8, SEQ ID NO: 56 and SEQ ID NO: 57 and at leastone light chain CDR selected from the group of SEQ ID NO: 59, SEQ ID NO:60, SEQ ID NO: 65.

(iii) a third antigen binding moiety capable of specific binding toFolate Receptor 1 (FolR1) comprising at least one heavy chaincomplementarity determining region (CDR) selected from the groupconsisting of SEQ ID NO: 8, SEQ ID NO: 56 and SEQ ID NO: 57 and at leastone light chain CDR selected from the group of SEQ ID NO: 59, SEQ ID NO:60, SEQ ID NO: 65.

In one such embodiment the CD3 antigen binding moiety comprises theheavy chain CDR1 of SEQ ID NO: 37, the heavy chain CDR2 of SEQ ID NO:38, the heavy chain CDR3 of SEQ ID NO:39, the light chain CDR1 of SEQ IDNO: 32, the light chain CDR2 of SEQ ID NO: 33, and the light chain CDR3of SEQ ID NO:34 and the FolR1 antigen binding moiety comprises the heavychain CDR1 of SEQ ID NO: 8, the heavy chain CDR2 of SEQ ID NO: 56, theheavy chain CDR3 of SEQ ID NO:57, the light chain CDR1 of SEQ ID NO: 59,the light chain CDR2 of SEQ ID NO: 60, and the light chain CDR3 of SEQID NO:65.

In one embodiment, the second antigen binding moiety and the thirdantigen binding moiety are both a conventional Fab molecule.

In one embodiment the T cell activating bispecific antigen bindingmolecule comprises

(i) a first antigen binding moiety which is a crossover Fab moleculecapable of specific binding to CD3 comprising a variable heavy chaincomprising an amino acid sequence of SEQ ID NO: 36 and a variable lightchain comprising an amino acid sequence of SEQ ID NO: 31.

(ii) a second antigen binding moiety which is a Fab molecule capable ofspecific binding to Folate Receptor 1 (FolR1) comprising a variableheavy chain comprising an amino acid sequence of SEQ ID NO: 55 and avariable light chain comprising an amino acid sequence of SEQ ID NO: 64.

(iii) a third antigen binding moiety which is a Fab molecule capable ofspecific binding to Folate Receptor 1 (FolR1) comprising a variableheavy chain comprising an amino acid sequence of SEQ ID NO: 55 and avariable light chain comprising an amino acid sequence of SEQ ID NO: 64.

In one embodiment, the second antigen binding moiety and the thirdantigen binding moiety are both a conventional Fab molecule.

In one embodiment the T cell activating bispecific antigen bindingmolecule comprises

(i) a first antigen binding moiety which is a crossover Fab moleculecapable of specific binding to CD3, comprising at least one heavy chaincomplementarity determining region (CDR) selected from the groupconsisting of SEQ ID NO: 37, SEQ ID NO: 38 and SEQ ID NO: 39 and atleast one light chain CDR selected from the group of SEQ ID NO: 32, SEQID NO: 33, SEQ ID NO: 34;

(ii) a second antigen binding moiety capable of specific binding toFolate Receptor 1 (FolR1) comprising at least one heavy chaincomplementarity determining region (CDR) selected from the groupconsisting of SEQ ID NO: 8, SEQ ID NO: 9 and SEQ ID NO: 50 and at leastone light chain CDR selected from the group of SEQ ID NO: 52, SEQ ID NO:53, SEQ ID NO: 54.

In one such embodiment the CD3 antigen binding moiety comprises theheavy chain CDR1 of SEQ ID NO: 37, the heavy chain CDR2 of SEQ ID NO:38, the heavy chain CDR3 of SEQ ID NO:39, the light chain CDR1 of SEQ IDNO: 32, the light chain CDR2 of SEQ ID NO: 33, and the light chain CDR3of SEQ ID NO:34 and the FolR1 antigen binding moiety comprises the heavychain CDR1 of SEQ ID NO: 8, the heavy chain CDR2 of SEQ ID NO: 9, theheavy chain CDR3 of SEQ ID NO:50, the light chain CDR1 of SEQ ID NO: 52,the light chain CDR2 of SEQ ID NO: 53, and the light chain CDR3 of SEQID NO:54.

In one embodiment, the second antigen binding moiety is a conventionalFab molecule. In one embodiment, the second antigen binding moiety is acrossover Fab molecule.

In one embodiment the T cell activating bispecific antigen bindingmolecule comprises

(i) a first antigen binding moiety which is a crossover Fab moleculecapable of specific binding to CD3 comprising a variable heavy chaincomprising an amino acid sequence of SEQ ID NO: 36 and a variable lightchain comprising an amino acid sequence of SEQ ID NO: 31.

(ii) a second antigen binding moiety which is a Fab molecule capable ofspecific binding to Folate Receptor 1 (FolR1) comprising a variableheavy chain comprising an amino acid sequence of SEQ ID NO: 49 and avariable light chain comprising an amino acid sequence of SEQ ID NO: 51.

In one embodiment, the second antigen binding moiety is a conventionalFab molecule. In one embodiment, the second antigen binding moiety is acrossover Fab molecule.

In a further embodiment, the antigen binding moiety that is specific forFolR1 comprises a heavy chain variable region sequence that is at leastabout 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO:49 and alight chain variable region sequence that is at least about 95%, 96%,97%, 98%, 99% or 100% identical to SEQ ID NO: 51 or variants thereofthat retain functionality.

In one embodiment the T cell activating bispecific antigen bindingmolecule comprises a polypeptide sequence that is at least about 95%,96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 36, a polypeptidesequence that is at least about 95%, 96%, 97%, 98%, 99% or 100%identical to SEQ ID NO: 31, a polypeptide sequence that is at leastabout 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO:49, and apolypeptide sequence that is at least about 95%, 96%, 97%, 98%, 99% or100% identical to SEQ ID NO: 51.

In one embodiment the T cell activating bispecific antigen bindingmolecule additionally comprises

-   -   (iii) a third antigen binding moiety capable of specific binding        to FolR1.

In one embodiment, the third antigen binding moiety is a conventionalFab molecule. In one embodiment, the second antigen binding moiety is acrossover Fab molecule.

In one such embodiment the second and third antigen binding moietycapable of specific binding to FolR1 comprise identical heavy chaincomplementarity determining region (CDR) and light chain CDR sequences.In one such embodiment the third antigen binding moiety is identical tothe second antigen binding moiety.

In one embodiment the T cell activating bispecific antigen bindingmolecule comprises

(i) a first antigen binding moiety which is a crossover Fab moleculecapable of specific binding to CD3, comprising at least one heavy chaincomplementarity determining region (CDR) selected from the groupconsisting of SEQ ID NO: 37, SEQ ID NO: 38 and SEQ ID NO: 39 and atleast one light chain CDR selected from the group of SEQ ID NO: 32, SEQID NO: 33, SEQ ID NO: 34;

(ii) a second antigen binding moiety capable of specific binding toFolate Receptor 1 (FolR1) comprising at least one heavy chaincomplementarity determining region (CDR) selected from the groupconsisting of SEQ ID NO: 8, SEQ ID NO: 9 and SEQ ID NO: 49 and at leastone light chain CDR selected from the group of SEQ ID NO: 52, SEQ ID NO:53, SEQ ID NO: 54.

(iii) a third antigen binding moiety capable of specific binding toFolate Receptor 1 (FolR1) comprising at least one heavy chaincomplementarity determining region (CDR) selected from the groupconsisting of SEQ ID NO: 8, SEQ ID NO: 9 and SEQ ID NO: 50 and at leastone light chain CDR selected from the group of SEQ ID NO: 52, SEQ ID NO:53, SEQ ID NO: 54.

In one such embodiment the CD3 antigen binding moiety comprises theheavy chain CDR1 of SEQ ID NO: 37, the heavy chain CDR2 of SEQ ID NO:38, the heavy chain CDR3 of SEQ ID NO:39, the light chain CDR1 of SEQ IDNO: 32, the light chain CDR2 of SEQ ID NO: 33, and the light chain CDR3of SEQ ID NO:34 and the FolR1 antigen binding moiety comprises the heavychain CDR1 of SEQ ID NO: 8, the heavy chain CDR2 of SEQ ID NO: 9, theheavy chain CDR3 of SEQ ID NO:50, the light chain CDR1 of SEQ ID NO: 52,the light chain CDR2 of SEQ ID NO: 53, and the light chain CDR3 of SEQID NO:54.

In one embodiment, the second antigen binding moiety and the thirdantigen binding moiety are both a conventional Fab molecule.

In one embodiment the T cell activating bispecific antigen bindingmolecule comprises

(i) a first antigen binding moiety which is a crossover Fab moleculecapable of specific binding to CD3 comprising a variable heavy chaincomprising an amino acid sequence of SEQ ID NO: 36 and a variable lightchain comprising an amino acid sequence of SEQ ID NO: 31.

(ii) a second antigen binding moiety which is a Fab molecule capable ofspecific binding to Folate Receptor 1 (FolR1) comprising a variableheavy chain comprising an amino acid sequence of SEQ ID NO: 49 and avariable light chain comprising an amino acid sequence of SEQ ID NO: 51.

(iii) a third antigen binding moiety which is a Fab molecule capable ofspecific binding to Folate Receptor 1 (FolR1) comprising a variableheavy chain comprising an amino acid sequence of SEQ ID NO: 49 and avariable light chain comprising an amino acid sequence of SEQ ID NO: 51.

In one embodiment, the second antigen binding moiety and the thirdantigen binding moiety are both a conventional Fab molecule.

Thus, in one embodiment, the invention relates to bispecific moleculeswherein two binding moieties confer specific binding to FolR1 and onebinding moiety confers specificity to the T cell activating antigen CD3.One of the heavy chains is modified to ensure proper pairing of theheavy and light chains, thus eliminating the need for a common lightchain approach. The presence of two FolR1 binding sites enablesappropriate engagement with the target antigen FolR1 and the activationof T cells.

The components of the T cell activating bispecific antigen bindingmolecule can be fused to each other in a variety of configurations.Exemplary configurations are depicted in FIGS. 1A-I and are furtherdescribed below.

In some embodiments, said T cell activating bispecific antigen bindingmolecule further comprises an Fc domain composed of a first and a secondsubunit capable of stable association. Below exemplary embodiments of Tcell activating bispecific antigen binding molecule comprising an Fcdomain are described.

T Cell Activating Bispecific Antigen Binding Molecule Formats

As depicted above and in FIGS. 1A-I, in one embodiment the T cellactivating bispecific antigen binding molecules comprise at least twoFab fragments having identical light chains (VLCL) and having differentheavy chains (VHCL) which confer the specificities to two differentantigens, i.e. one Fab fragment is capable of specific binding to a Tcell activating antigen CD3 and the other Fab fragment is capable ofspecific binding to the target cell antigen FolR1.

In another embodiment the T cell activating bispecific antigen bindingmolecule comprises at least two antigen binding moieties (Fabmolecules), one of which is a crossover Fab molecule and one of which isa conventional Fab molecule. In one such embodiment the first antigenbinding moiety capable of specific binding to CD3 is a crossover Fabmolecule and the second antigen binding moiety capable of specificbinding to FolR is a conventional Fab molecule.

These components of the T cell activating bispecific antigen bindingmolecule can be fused to each other in a variety of configurations.Exemplary configurations are depicted in FIGS. 1A-I.

In some embodiments, the first and second antigen binding moiety areeach fused at the C-terminus of the Fab heavy chain to the N-terminus ofthe first or the second subunit of the Fc domain. In a specific suchembodiment, the T cell activating bispecific antigen binding moleculeessentially consists of a first and a second antigen binding moiety, anFc domain composed of a first and a second subunit, and optionally oneor more peptide linkers, wherein the first and second antigen bindingmoiety are each fused at the C-terminus of the Fab heavy chain to theN-terminus of the first or the second subunit of the Fc domain. In onesuch embodiment the first and second antigen binding moiety both are Fabfragments and have identical light chains (VLCL). In another suchembodiment the first antigen binding moiety capable of specific bindingto CD3 is a crossover Fab molecule and the second antigen binding moietycapable of specific binding to FolR is a conventional Fab molecule.

In one embodiment, the second antigen binding moiety is fused at theC-terminus of the Fab heavy chain to the N-terminus of the first or thesecond subunit of the Fc domain and the first antigen binding moiety isfused at the C-terminus of the Fab heavy chain to the N-terminus of theFab heavy chain of the second antigen binding moiety. In a specific suchembodiment, the T cell activating bispecific antigen binding moleculeessentially consists of a first and a second antigen binding moiety, anFc domain composed of a first and a second subunit, and optionally oneor more peptide linkers, wherein the first antigen binding moiety isfused at the C-terminus of the Fab heavy chain to the N-terminus of theFab heavy chain of the second antigen binding moiety, and the secondantigen binding moiety is fused at the C-terminus of the Fab heavy chainto the N-terminus of the first or the second subunit of the Fc domain.In one such embodiment the first and second antigen binding moiety bothare Fab fragments and have identical light chains (VLCL). In anothersuch embodiment the first antigen binding moiety capable of specificbinding to CD3 is a crossover Fab molecule and the second antigenbinding moiety capable of specific binding to FolR is a conventional Fabmolecule. Optionally, the Fab light chain of the first antigen bindingmoiety and the Fab light chain of the second antigen binding moiety mayadditionally be fused to each other.

In other embodiments, the first antigen binding moiety is fused at theC-terminus of the Fab heavy chain to the N-terminus of the first orsecond subunit of the Fc domain. In a particular such embodiment, thesecond antigen binding moiety is fused at the C-terminus of the Fabheavy chain to the N-terminus of the Fab heavy chain of the firstantigen binding moiety. In a specific such embodiment, the T cellactivating bispecific antigen binding molecule essentially consists of afirst and a second antigen binding moiety, an Fc domain composed of afirst and a second subunit, and optionally one or more peptide linkers,wherein the second antigen binding moiety is fused at the C-terminus ofthe Fab heavy chain to the N-terminus of the Fab heavy chain of thefirst antigen binding moiety, and the first antigen binding moiety isfused at the C-terminus of the Fab heavy chain to the N-terminus of thefirst or the second subunit of the Fc domain. In one such embodiment thefirst and second antigen binding moiety both are Fab fragments and haveidentical light chains (VLCL). In another such embodiment the firstantigen binding moiety capable of specific binding to CD3 is a crossoverFab molecule and the second antigen binding moiety capable of specificbinding to FolR is a conventional Fab molecule. Optionally, the Fablight chain of the first antigen binding moiety and the Fab light chainof the second antigen binding moiety may additionally be fused to eachother.

The antigen binding moieties may be fused to the Fc domain or to eachother directly or through a peptide linker, comprising one or more aminoacids, typically about 2-20 amino acids. Peptide linkers are known inthe art and are described herein. Suitable, non-immunogenic peptidelinkers include, for example, (G₄S)_(n) (SEQ ID NO: 300), (SG₄)_(n) (SEQID NO: 301), (G₄S)_(n) (SEQ ID NO: 300) or G₄(SG₄)_(n) (SEQ ID NO: 302)peptide linkers. “n” is generally a number between 1 and 10, typicallybetween 2 and 4. A particularly suitable peptide linker for fusing theFab light chains of the first and the second antigen binding moiety toeach other is (G₄S)₂ (SEQ ID NO: 303). An exemplary peptide linkersuitable for connecting the Fab heavy chains of the first and the secondantigen binding moiety is EPKSC(D)-(G₄S)₂ (SEQ ID NOS 304 and 305).Additionally, linkers may comprise (a portion of) an immunoglobulinhinge region. Particularly where an antigen binding moiety is fused tothe N-terminus of an Fc domain subunit, it may be fused via animmunoglobulin hinge region or a portion thereof, with or without anadditional peptide linker.

It has been found by the inventors of the present invention that T cellactivating bispecific antigen binding molecule comprising two bindingmoieties specific for the target cell antigen FolR have superiorcharacteristics compared to T cell activating bispecific antigen bindingmolecule comprising only one binding moiety specific for the target cellantigen FolR.

Accordingly, in certain embodiments, the T cell activating bispecificantigen binding molecule of the invention further comprises a thirdantigen binding moiety which is a Fab molecule capable of specificbinding to FolR. In one such embodiment the second and third antigenbinding moiety capable of specific binding to FolR1 comprise identicalheavy chain complementarity determining region (CDR) and light chain CDRsequences. In one such embodiment the third antigen binding moiety isidentical to the second antigen binding moiety (i.e. they comprise thesame amino acid sequences).

In one embodiment, the first and second antigen binding moiety are eachfused at the C-terminus of the Fab heavy chain to the N-terminus of thefirst or second subunit of the Fc domain and the third antigen bindingmoiety is fused at the C-terminus of the Fab heavy chain to theN-terminus to the N-terminus of the Fab heavy chain of the first antigenbinding moiety. In a specific such embodiment, the T cell activatingbispecific antigen binding molecule essentially consists of a first, asecond and a third antigen binding moiety, an Fc domain composed of afirst and a second subunit, and optionally one or more peptide linkers,wherein the first and second antigen binding moiety are each fused atthe C-terminus of the Fab heavy chain to the N-terminus of the firstsubunit of the Fc domain and the third antigen binding moiety is fusedat the C-terminus of the Fab heavy chain to the N-terminus of the Fabheavy chain of the first antigen binding moiety. In one such embodimentthe first, second and third antigen binding moiety are conventional Fabfragments and have identical light chains (VLCL). In another suchembodiment the first antigen binding moiety capable of specific bindingto CD3 is a crossover Fab molecule and the second and third antigenbinding moiety capable of specific binding to FolR is a conventional Fabmolecule. Optionally, the Fab light chain of the first antigen bindingmoiety and the Fab light chain of the third antigen binding moiety mayadditionally be fused to each other.

Accordingly, in certain embodiments, the T cell activating bispecificantigen binding molecule of the invention comprises five polypeptidechains that form a first, a second and a third antigen binding moietywherein the first antigen binding moiety is capable of binding CD3 andthe second and the third antigen binding moiety each are capable ofbinding Folate Receptor 1 (FolR1). The first and the second polypeptidechain comprise, in amino (N)-terminal to carboxyl (C)-terminaldirection, a first light chain variable domain (VLD1) and a first lightchain constant domain (CLD1). The third polypeptide chain comprises, inN-terminal to C-terminal direction, second light chain variable domain(VLD2) and a second heavy chain constant domain 1 (CH1D2). The fourthpolypeptide chain comprises, in N-terminal to C-terminal direction, afirst heavy chain variable domain (VHD1), a first heavy chain constantdomain 1 (CH1D1), a first heavy chain constant domain 2 (CH2D1) and afirst heavy chain constant domain 3 (CH3D1). The fifth polypeptide chaincomprises VHD1, CH1D1, a second heavy chain variable domain (VHD2), asecond light chain constant domain (CLD2), a second heavy chain constantdomain 2 (CH2D2) and a second heavy chain constant domain 3 (CH3D2). Thethird polypeptide chain and VHD2 and CLD2 of the fifth polypeptide chainform the first antigen binding moiety capable of binding CD3. The secondpolypeptide chain and VHD1 and CH1D1 of the fifth polypeptide chain formthe third binding moiety capable of binding to FolR1. The firstpolypeptide chain and VHD1 and CH1D1 of the fourth polypeptide chainform the second binding moiety capable of binding to FolR1.

In another embodiment, the second and the third antigen binding moietyare each fused at the C-terminus of the Fab heavy chain to theN-terminus of the first or second subunit of the Fc domain, and thefirst antigen binding moiety is fused at the C-terminus of the Fab heavychain to the N-terminus of the Fab heavy chain of the second antigenbinding moiety. In a specific such embodiment, the T cell activatingbispecific antigen binding molecule essentially consists of a first, asecond and a third antigen binding moiety, an Fc domain composed of afirst and a second subunit, and optionally one or more peptide linkers,wherein the second and third antigen binding moiety are each fused atthe C-terminus of the Fab heavy chain to the N-terminus of the firstsubunit of the Fc domain and the first antigen binding moiety is fusedat the C-terminus of the Fab heavy chain to the N-terminus of the Fabheavy chain of the third antigen binding moiety. In one such embodimentthe first, second and third antigen binding moiety are conventional Fabfragments and have identical light chains (VLCL). In another suchembodiment the first antigen binding moiety capable of specific bindingto CD3 is a crossover Fab molecule and the second and third antigenbinding moiety capable of specific binding to FolR is a conventional Fabmolecule. Optionally, the Fab light chain of the first antigen bindingmoiety and the Fab light chain of the second antigen binding moiety mayadditionally be fused to each other.

The antigen binding moieties may be fused to the Fc domain directly orthrough a peptide linker. In a particular embodiment the antigen bindingmoieties are each fused to the Fc domain through an immunoglobulin hingeregion. In a specific embodiment, the immunoglobulin hinge region is ahuman IgG₁ hinge region.

In one embodiment the first and the second antigen binding moiety andthe Fc domain are part of an immunoglobulin molecule. In a particularembodiment the immunoglobulin molecule is an IgG class immunoglobulin.In an even more particular embodiment the immunoglobulin is an IgG₁subclass immunoglobulin. In another embodiment the immunoglobulin is anIgG₄ subclass immunoglobulin. In a further particular embodiment theimmunoglobulin is a human immunoglobulin. In other embodiments theimmunoglobulin is a chimeric immunoglobulin or a humanizedimmunoglobulin.

In a particular embodiment said T cell activating bispecific antigenbinding molecule the first and the second antigen binding moiety and theFc domain are part of an immunoglobulin molecule, and the third antigenbinding moiety is fused at the C-terminus of the Fab heavy chain to theN-terminus of the Fab heavy chain of the first antigen binding moiety,wherein the first, second and third antigen binding moiety areconventional Fab fragments and have identical light chains (VLCL),wherein the first antigen binding moiety capable of specific binding toCD3 comprises at least one heavy chain complementarity determiningregion (CDR) selected from the group consisting of SEQ ID NO: 37, SEQ IDNO: 38 and SEQ ID NO: 39 and at least one light chain CDR selected fromthe group of SEQ ID NO: 32, SEQ ID NO: 33 and SEQ ID NO: 34; and thesecond and the third antigen binding moiety capable of specific bindingto FolR1 comprise at least one heavy chain complementarity determiningregion (CDR) selected from the group consisting of SEQ ID NO: 16, SEQ IDNO: 17 and SEQ ID NO: 18 and at least one light chain CDR selected fromthe group of SEQ ID NO: 32, SEQ ID NO: 33 and SEQ ID NO: 34.

In a particular embodiment said T cell activating bispecific antigenbinding molecule the first and the second antigen binding moiety and theFc domain are part of an immunoglobulin molecule, and the third antigenbinding moiety is fused at the C-terminus of the Fab heavy chain to theN-terminus of the Fab heavy chain of the first antigen binding moiety,wherein the first, second and third antigen binding moiety areconventional Fab fragments and have identical light chains (VLCL),wherein the first antigen binding moiety capable of specific binding toCD3 comprises a variable heavy chain comprising a sequence of SEQ ID NO:36, a variable light chain comprising a sequence of SEQ ID NO: 31; andthe second and the third antigen binding moiety capable of specificbinding to FolR1 comprise a variable heavy chain comprising a sequenceof SEQ ID NO: 15, a variable light chain comprising a sequence of SEQ IDNO: 31.

In a particular embodiment said T cell activating bispecific antigenbinding molecule the first and the second antigen binding moiety and theFc domain are part of an immunoglobulin molecule, and the third antigenbinding moiety is fused at the C-terminus of the Fab heavy chain to theN-terminus of the Fab heavy chain of the first antigen binding moietyand the first antigen binding moiety capable of specific binding to CD3is a crossover Fab molecule wherein either the variable or the constantregions of the Fab light chain and the Fab heavy chain are exchanged,comprising at least one heavy chain complementarity determining region(CDR) selected from the group consisting of SEQ ID NO: 37, SEQ ID NO: 38and SEQ ID NO: 39 and at least one light chain CDR selected from thegroup of SEQ ID NO: 32, SEQ ID NO: 33 and SEQ ID NO: 34; and the secondand the third antigen binding moiety capable of specific binding toFolR1 comprise at least one heavy chain complementarity determiningregion (CDR) selected from the group consisting of SEQ ID NO: 8, SEQ IDNO: 56 and SEQ ID NO: 57 and at least one light chain CDR selected fromthe group of SEQ ID NO: 59, SEQ ID NO: 60 and SEQ ID NO: 65.

In a particular embodiment said T cell activating bispecific antigenbinding molecule the first and the second antigen binding moiety and theFc domain are part of an immunoglobulin molecule, and the third antigenbinding moiety is fused at the C-terminus of the Fab heavy chain to theN-terminus of the Fab heavy chain of the first antigen binding moietyand the first antigen binding moiety capable of specific binding to CD3is a crossover Fab molecule wherein either the variable or the constantregions of the Fab light chain and the Fab heavy chain are exchanged,wherein the first antigen binding moiety capable of specific binding toCD3 comprises a variable heavy chain comprising a sequence of SEQ ID NO:36, a variable light chain comprising a sequence of SEQ ID NO: 31; andthe second and the third antigen binding moiety capable of specificbinding to FolR1 comprise a variable heavy chain comprising a sequenceof SEQ ID NO: 55, a variable light chain comprising a sequence of SEQ IDNO: 65.

In one embodiment the T cell activating bispecific antigen bindingmolecule is monovalent for each antigen. In a particular embodiment theT cell activating bispecific antigen binding molecule can bind to humanCD3 and human folate receptor alpha (FolR1) and was made withoutemploying a hetero-dimerization approach, such as, e.g., knob-into-holetechnology. For example, the molecule can be produced by employing acommon light chain library and CrossMab technology. In a particularembodiment, The variable region of the CD3 binder is fused to the CH1domain of a standard human IgG1 antibody to form the VLVH crossedmolecule (fused to Fc) which is common for both specificities. Togenerate the crossed counterparts (VHCL), a CD3 specific variable heavychain domain is fused to a constant human λ light chain whereas avariable heavy chain domain specific for human FolR1 (e.g., isolatedfrom a common light chain library) is fused to a constant human κ lightchain. The resulting desired molecule with correctly paired chainscomprises both kappa and lambda light chains or fragments thereof.Consequently, this desired bispecific molecule species can be purifiedfrom mispaired or homodimeric species with sequential purification stepsselecting for kappa and lambda light chain, in either sequence. In oneparticular embodiment, purification of the desired bispecific antibodyemploys subsequent purification steps with KappaSelect andLambdaFabSelect columns (GE Healthcare) to remove undesired homodimericantibodies.

Fc Domain

The Fc domain of the T cell activating bispecific antigen bindingmolecule consists of a pair of polypeptide chains comprising heavy chaindomains of an immunoglobulin molecule. For example, the Fc domain of animmunoglobulin G (IgG) molecule is a dimer, each subunit of whichcomprises the CH2 and CH3 IgG heavy chain constant domains. The twosubunits of the Fc domain are capable of stable association with eachother. In one embodiment the T cell activating bispecific antigenbinding molecule of the invention comprises not more than one Fc domain.

In one embodiment according the invention the Fc domain of the T cellactivating bispecific antigen binding molecule is an IgG Fc domain. In aparticular embodiment the Fc domain is an IgG₁ Fc domain. In anotherembodiment the Fc domain is an IgG₄ Fc domain. In a more specificembodiment, the Fc domain is an IgG₄ Fc domain comprising an amino acidsubstitution at position S228 (Kabat numbering), particularly the aminoacid substitution S228P. This amino acid substitution reduces in vivoFab arm exchange of IgG₄ antibodies (see Stubenrauch et al., DrugMetabolism and Disposition 38, 84-91 (2010)). In a further particularembodiment the Fc domain is human. An exemplary sequence of a human IgG₁Fc region is given in SEQ ID NO:245.

Fc Domain Modifications Promoting Heterodimerization

T cell activating bispecific antigen binding molecules according to theinvention comprise different antigen binding moieties, fused to one orthe other of the two subunits of the Fc domain, thus the two subunits ofthe Fc domain are typically comprised in two non-identical polypeptidechains. Recombinant co-expression of these polypeptides and subsequentdimerization leads to several possible combinations of the twopolypeptides. To improve the yield and purity of T cell activatingbispecific antigen binding molecules in recombinant production, it willthus be advantageous to introduce in the Fc domain of the T cellactivating bispecific antigen binding molecule a modification promotingthe association of the desired polypeptides.

Accordingly, in particular embodiments the Fc domain of the T cellactivating bispecific antigen binding molecule according to theinvention comprises a modification promoting the association of thefirst and the second subunit of the Fc domain. The site of mostextensive protein-protein interaction between the two subunits of ahuman IgG Fc domain is in the CH3 domain of the Fc domain. Thus, in oneembodiment said modification is in the CH3 domain of the Fc domain.

In a specific embodiment said modification is a so-called“knob-into-hole” modification, comprising a “knob” modification in oneof the two subunits of the Fc domain and a “hole” modification in theother one of the two subunits of the Fc domain.

The knob-into-hole technology is described e.g. in U.S. Pat. No.5,731,168; U.S. Pat. No. 7,695,936; Ridgway et al., Prot Eng 9, 617-621(1996) and Carter, J Immunol Meth 248, 7-15 (2001). Generally, themethod involves introducing a protuberance (“knob”) at the interface ofa first polypeptide and a corresponding cavity (“hole”) in the interfaceof a second polypeptide, such that the protuberance can be positioned inthe cavity so as to promote heterodimer formation and hinder homodimerformation. Protuberances are constructed by replacing small amino acidside chains from the interface of the first polypeptide with larger sidechains (e.g. tyrosine or tryptophan). Compensatory cavities of identicalor similar size to the protuberances are created in the interface of thesecond polypeptide by replacing large amino acid side chains withsmaller ones (e.g. alanine or threonine). Accordingly, in a particularembodiment, in the CH3 domain of the first subunit of the Fc domain ofthe T cell activating bispecific antigen binding molecule an amino acidresidue is replaced with an amino acid residue having a larger sidechain volume, thereby generating a protuberance within the CH3 domain ofthe first subunit which is positionable in a cavity within the CH3domain of the second subunit, and in the CH3 domain of the secondsubunit of the Fc domain an amino acid residue is replaced with an aminoacid residue having a smaller side chain volume, thereby generating acavity within the CH3 domain of the second subunit within which theprotuberance within the CH3 domain of the first subunit is positionable.

The protuberance and cavity can be made by altering the nucleic acidencoding the polypeptides, e.g. by site-specific mutagenesis, or bypeptide synthesis.

In a specific embodiment, in the CH3 domain of the first subunit of theFc domain the threonine residue at position 366 is replaced with atryptophan residue (T366W), and in the CH3 domain of the second subunitof the Fc domain the tyrosine residue at position 407 is replaced with avaline residue (Y407V). In one embodiment, in the second subunit of theFc domain additionally the threonine residue at position 366 is replacedwith a serine residue (T366S) and the leucine residue at position 368 isreplaced with an alanine residue (L368A).

In yet a further embodiment, in the first subunit of the Fc domainadditionally the serine residue at position 354 is replaced with acysteine residue (S354C), and in the second subunit of the Fc domainadditionally the tyrosine residue at position 349 is replaced by acysteine residue (Y349C). Introduction of these two cysteine residuesresults in formation of a disulfide bridge between the two subunits ofthe Fc domain, thus further stabilizing the dimer (Carter, J ImmunolMethods 248, 7-15 (2001)).

In a particular embodiment the antigen binding moiety capable of bindingto CD3 is fused (optionally via the antigen binding moiety capable ofbinding to FolR1 on a target cell antigen) to the first subunit of theFc domain (comprising the “knob” modification). Without wishing to bebound by theory, fusion of the antigen binding moiety capable of bindingto CD3 to the knob-containing subunit of the Fc domain will (further)minimize the generation of antigen binding molecules comprising twoantigen binding moieties capable of binding to CD3 (steric clash of twoknob-containing polypeptides).

In an alternative embodiment a modification promoting association of thefirst and the second subunit of the Fc domain comprises a modificationmediating electrostatic steering effects, e.g. as described in PCTpublication WO 2009/089004. Generally, this method involves replacementof one or more amino acid residues at the interface of the two Fc domainsubunits by charged amino acid residues so that homodimer formationbecomes electrostatically unfavorable but heterodimerizationelectrostatically favorable.

Fc Domain Modifications Abolishing Fc Receptor Binding and/or EffectorFunction

The Fc domain confers to the T cell activating bispecific antigenbinding molecule favorable pharmacokinetic properties, including a longserum half-life which contributes to good accumulation in the targettissue and a favorable tissue-blood distribution ratio. At the same timeit may, however, lead to undesirable targeting of the T cell activatingbispecific antigen binding molecule to cells expressing Fc receptorsrather than to the preferred antigen-bearing cells. Moreover, theco-activation of Fc receptor signaling pathways may lead to cytokinerelease which, in combination with the T cell activating properties andthe long half-life of the antigen binding molecule, results in excessiveactivation of cytokine receptors and severe side effects upon systemicadministration. Activation of (Fc receptor-bearing) immune cells otherthan T cells may even reduce efficacy of the T cell activatingbispecific antigen binding molecule due to the potential destruction ofT cells e.g. by NK cells.

Accordingly, in particular embodiments the Fc domain of the T cellactivating bispecific antigen binding molecules according to theinvention exhibits reduced binding affinity to an Fc receptor and/orreduced effector function, as compared to a native IgG₁ Fc domain. Inone such embodiment the Fc domain (or the T cell activating bispecificantigen binding molecule comprising said Fc domain) exhibits less than50%, preferably less than 20%, more preferably less than 10% and mostpreferably less than 5% of the binding affinity to an Fc receptor, ascompared to a native IgG₁ Fc domain (or a T cell activating bispecificantigen binding molecule comprising a native IgG₁ Fc domain), and/orless than 50%, preferably less than 20%, more preferably less than 10%and most preferably less than 5% of the effector function, as comparedto a native IgG₁ Fc domain domain (or a T cell activating bispecificantigen binding molecule comprising a native IgG₁ Fc domain). In oneembodiment, the Fc domain domain (or the T cell activating bispecificantigen binding molecule comprising said Fc domain) does notsubstantially bind to an Fc receptor and/or induce effector function. Ina particular embodiment the Fc receptor is an Fcγ receptor. In oneembodiment the Fc receptor is a human Fc receptor. In one embodiment theFc receptor is an activating Fc receptor. In a specific embodiment theFc receptor is an activating human Fcγ receptor, more specifically humanFcγRIIIa, FcγRI or FcγRIIa, most specifically human FcγRIIIa. In oneembodiment the effector function is one or more selected from the groupof CDC, ADCC, ADCP, and cytokine secretion. In a particular embodimentthe effector function is ADCC. In one embodiment the Fc domain domainexhibits substantially similar binding affinity to neonatal Fc receptor(FcRn), as compared to a native IgG₁ Fc domain domain. Substantiallysimilar binding to FcRn is achieved when the Fc domain (or the T cellactivating bispecific antigen binding molecule comprising said Fcdomain) exhibits greater than about 70%, particularly greater than about80%, more particularly greater than about 90% of the binding affinity ofa native IgG₁ Fc domain (or the T cell activating bispecific antigenbinding molecule comprising a native IgG₁ Fc domain) to FcRn.

In certain embodiments the Fc domain is engineered to have reducedbinding affinity to an Fc receptor and/or reduced effector function, ascompared to a non-engineered Fc domain. In particular embodiments, theFc domain of the T cell activating bispecific antigen binding moleculecomprises one or more amino acid mutation that reduces the bindingaffinity of the Fc domain to an Fc receptor and/or effector function.Typically, the same one or more amino acid mutation is present in eachof the two subunits of the Fc domain. In one embodiment the amino acidmutation reduces the binding affinity of the Fc domain to an Fcreceptor. In one embodiment the amino acid mutation reduces the bindingaffinity of the Fc domain to an Fc receptor by at least 2-fold, at least5-fold, or at least 10-fold. In embodiments where there is more than oneamino acid mutation that reduces the binding affinity of the Fc domainto the Fc receptor, the combination of these amino acid mutations mayreduce the binding affinity of the Fc domain to an Fc receptor by atleast 10-fold, at least 20-fold, or even at least 50-fold. In oneembodiment the T cell activating bispecific antigen binding moleculecomprising an engineered Fc domain exhibits less than 20%, particularlyless than 10%, more particularly less than 5% of the binding affinity toan Fc receptor as compared to a T cell activating bispecific antigenbinding molecule comprising a non-engineered Fc domain. In a particularembodiment the Fc receptor is an Fcγ receptor. In some embodiments theFc receptor is a human Fc receptor. In some embodiments the Fc receptoris an activating Fc receptor. In a specific embodiment the Fc receptoris an activating human Fcγ receptor, more specifically human FcγRIIIa,FcγRI or FcγRIIa, most specifically human FcγRIIIa. Preferably, bindingto each of these receptors is reduced. In some embodiments bindingaffinity to a complement component, specifically binding affinity toC1q, is also reduced. In one embodiment binding affinity to neonatal Fcreceptor (FcRn) is not reduced. Substantially similar binding to FcRn,i.e. preservation of the binding affinity of the Fc domain to saidreceptor, is achieved when the Fc domain (or the T cell activatingbispecific antigen binding molecule comprising said Fc domain) exhibitsgreater than about 70% of the binding affinity of a non-engineered formof the Fc domain (or the T cell activating bispecific antigen bindingmolecule comprising said non-engineered form of the Fc domain) to FcRn.The Fc domain, or T cell activating bispecific antigen binding moleculesof the invention comprising said Fc domain, may exhibit greater thanabout 80% and even greater than about 90% of such affinity. In certainembodiments the Fc domain of the T cell activating bispecific antigenbinding molecule is engineered to have reduced effector function, ascompared to a non-engineered Fc domain. The reduced effector functioncan include, but is not limited to, one or more of the following:reduced complement dependent cytotoxicity (CDC), reducedantibody-dependent cell-mediated cytotoxicity (ADCC), reducedantibody-dependent cellular phagocytosis (ADCP), reduced cytokinesecretion, reduced immune complex-mediated antigen uptake byantigen-presenting cells, reduced binding to NK cells, reduced bindingto macrophages, reduced binding to monocytes, reduced binding topolymorphonuclear cells, reduced direct signaling inducing apoptosis,reduced crosslinking of target-bound antibodies, reduced dendritic cellmaturation, or reduced T cell priming. In one embodiment the reducedeffector function is one or more selected from the group of reduced CDC,reduced ADCC, reduced ADCP, and reduced cytokine secretion. In aparticular embodiment the reduced effector function is reduced ADCC. Inone embodiment the reduced ADCC is less than 20% of the ADCC induced bya non-engineered Fc domain (or a T cell activating bispecific antigenbinding molecule comprising a non-engineered Fc domain).

In one embodiment the amino acid mutation that reduces the bindingaffinity of the Fc domain to an Fc receptor and/or effector function isan amino acid substitution. In one embodiment the Fc domain comprises anamino acid substitution at a position selected from the group of E233,L234, L235, N297, P331 and P329. In a more specific embodiment the Fcdomain comprises an amino acid substitution at a position selected fromthe group of L234, L235 and P329. In some embodiments the Fc domaincomprises the amino acid substitutions L234A and L235A. In one suchembodiment, the Fc domain is an IgG₁ Fc domain, particularly a humanIgG₁ Fc domain. In one embodiment the Fc domain comprises an amino acidsubstitution at position P329. In a more specific embodiment the aminoacid substitution is P329A or P329G, particularly P329G. In oneembodiment the Fc domain comprises an amino acid substitution atposition P329 and a further amino acid substitution at a positionselected from E233, L234, L235, N297 and P331. In a more specificembodiment the further amino acid substitution is E233P, L234A, L235A,L235E, N297A, N297D or P331S. In particular embodiments the Fc domaincomprises amino acid substitutions at positions P329, L234 and L235. Inmore particular embodiments the Fc domain comprises the amino acidmutations L234A, L235A and P329G (“P329G LALA”). In one such embodiment,the Fc domain is an IgG₁ Fc domain, particularly a human IgG₁ Fc domain.The “P329G LALA” combination of amino acid substitutions almostcompletely abolishes Fcγ receptor binding of a human IgG₁ Fc domain, asdescribed in PCT publication no. WO 2012/130831, incorporated herein byreference in its entirety. WO 2012/130831 also describes methods ofpreparing such mutant Fc domains and methods for determining itsproperties such as Fc receptor binding or effector functions.

IgG₄ antibodies exhibit reduced binding affinity to Fc receptors andreduced effector functions as compared to IgG₁ antibodies. Hence, insome embodiments the Fc domain of the T cell activating bispecificantigen binding molecules of the invention is an IgG₄ Fc domain,particularly a human IgG₄ Fc domain. In one embodiment the IgG₄ Fcdomain comprises amino acid substitutions at position S228, specificallythe amino acid substitution S228P. To further reduce its bindingaffinity to an Fc receptor and/or its effector function, in oneembodiment the IgG₄ Fc domain comprises an amino acid substitution atposition L235, specifically the amino acid substitution L235E. Inanother embodiment, the IgG₄ Fc domain comprises an amino acidsubstitution at position P329, specifically the amino acid substitutionP329G. In a particular embodiment, the IgG₄ Fc domain comprises aminoacid substitutions at positions S228, L235 and P329, specifically aminoacid substitutions S228P, L235E and P329G. Such IgG₄ Fc domain mutantsand their Fcγ receptor binding properties are described in PCTpublication no. WO 2012/130831, incorporated herein by reference in itsentirety.

In a particular embodiment the Fc domain exhibiting reduced bindingaffinity to an Fc receptor and/or reduced effector function, as comparedto a native IgG₁ Fc domain, is a human IgG₁ Fc domain comprising theamino acid substitutions L234A, L235A and optionally P329G, or a humanIgG₄ Fc domain comprising the amino acid substitutions S228P, L235E andoptionally P329G.

In certain embodiments N-glycosylation of the Fc domain has beeneliminated. In one such embodiment the Fc domain comprises an amino acidmutation at position N297, particularly an amino acid substitutionreplacing asparagine by alanine (N297A) or aspartic acid (N297D).

In addition to the Fc domains described hereinabove and in PCTpublication no. WO 2012/130831, Fc domains with reduced Fc receptorbinding and/or effector function also include those with substitution ofone or more of Fc domain residues 238, 265, 269, 270, 297, 327 and 329(U.S. Pat. No. 6,737,056). Such Fc mutants include Fc mutants withsubstitutions at two or more of amino acid positions 265, 269, 270, 297and 327, including the so-called “DANA” Fc mutant with substitution ofresidues 265 and 297 to alanine (U.S. Pat. No. 7,332,581).

Mutant Fc domains can be prepared by amino acid deletion, substitution,insertion or modification using genetic or chemical methods well knownin the art. Genetic methods may include site-specific mutagenesis of theencoding DNA sequence, PCR, gene synthesis, and the like. The correctnucleotide changes can be verified for example by sequencing.

Binding to Fc receptors can be easily determined e.g. by ELISA, or bySurface Plasmon Resonance (SPR) using standard instrumentation such as aBIAcore instrument (GE Healthcare), and Fc receptors such as may beobtained by recombinant expression. A suitable such binding assay isdescribed herein. Alternatively, binding affinity of Fc domains or cellactivating bispecific antigen binding molecules comprising an Fc domainfor Fc receptors may be evaluated using cell lines known to expressparticular Fc receptors, such as human NK cells expressing FcγIIIareceptor.

Effector function of an Fc domain, or a T cell activating bispecificantigen binding molecule comprising an Fc domain, can be measured bymethods known in the art. A suitable assay for measuring ADCC isdescribed herein. Other examples of in vitro assays to assess ADCCactivity of a molecule of interest are described in U.S. Pat. No.5,500,362; Hellstrom et al. Proc Natl Acad Sci USA 83, 7059-7063 (1986)and Hellstrom et al., Proc Natl Acad Sci USA 82, 1499-1502 (1985); U.S.Pat. No. 5,821,337; Bruggemann et al., J Exp Med 166, 1351-1361 (1987).Alternatively, non-radioactive assays methods may be employed (see, forexample, ACTI™ non-radioactive cytotoxicity assay for flow cytometry(CellTechnology, Inc. Mountain View, Calif.); and CytoTox 96®non-radioactive cytotoxicity assay (Promega, Madison, Wis.)). Usefuleffector cells for such assays include peripheral blood mononuclearcells (PBMC) and Natural Killer (NK) cells. Alternatively, oradditionally, ADCC activity of the molecule of interest may be assessedin vivo, e.g. in a animal model such as that disclosed in Clynes et al.,Proc Natl Acad Sci USA 95, 652-656 (1998).

In some embodiments, binding of the Fc domain to a complement component,specifically to C1q, is reduced. Accordingly, in some embodimentswherein the Fc domain is engineered to have reduced effector function,said reduced effector function includes reduced CDC. C1q binding assaysmay be carried out to determine whether the T cell activating bispecificantigen binding molecule is able to bind C1q and hence has CDC activity.See e.g., C1q and C3c binding ELISA in WO 2006/029879 and WO2005/100402. To assess complement activation, a CDC assay may beperformed (see, for example, Gazzano-Santoro et al., J Immunol Methods202, 163 (1996); Cragg et al., Blood 101, 1045-1052 (2003); and Craggand Glennie, Blood 103, 2738-2743 (2004)).

Biological Properties and Functional Characteristics of T CellActivating Bispecific Antigen Binding Molecules

One of skill in the art can appreciate the advantageous efficiency of amolecule that selectively distinguishes between cancerous andnon-cancerous, healthy cells. One way to accomplish this goal is byappropriate target selection. Markers expressed exclusively on tumorcells can be employed to selectively target effector molecules or cellsto tumor cells while sparing normal cells that do not express suchmarker. However, in some instances, so called tumor cell markers arealso expressed in normal tissue, albeit at lower levels. This expressionin normal tissue raises the possibility of toxicity. Thus, there was aneed in the art for molecules that can more selectively target tumorcells. The invention described herein provides for T cell activatingbispecific antigen binding molecules that selectively targetFolR1-positive tumor cells and not normal, non-cancerous cells thatexpress FolR1 at low levels or not at all. In one embodiment, the T cellactivating bispecific antigen binding molecule comprises at least two,preferably two, FolR1 binding moieties of relatively low affinity thatconfer an avidity effect which allows for differentiation between highand low FolR1 expressing cells. Because tumor cells express FolR1 athigh or intermediate levels, this embodiment of the inventionselectively binds to, and/or induces killing of, tumor cells and notnormal, non-cancerous cells that express FolR1 at low levels or not atall. In one embodiment, the T cell activating bispecific antigen bindingmolecule is in the 2+1 inverted format. In one embodiment, the T cellactivating bispecific antigen binding molecule induces T cell mediatedkilling of FolR1-positive tumor cells and not non-tumor cells andcomprises a CD3 antigen binding moiety that comprises the heavy chainCDR1 of SEQ ID NO: 37, the heavy chain CDR2 of SEQ ID NO: 38, the heavychain CDR3 of SEQ ID NO:39, the light chain CDR1 of SEQ ID NO: 32, thelight chain CDR2 of SEQ ID NO: 33, and the light chain CDR3 of SEQ IDNO:34 and two FolR1 antigen binding moieties that each comprise theheavy chain CDR1 of SEQ ID NO: 8, the heavy chain CDR2 of SEQ ID NO: 9,the heavy chain CDR3 of SEQ ID NO:50, the light chain CDR1 of SEQ ID NO:52, the light chain CDR2 of SEQ ID NO: 53, and the light chain CDR3 ofSEQ ID NO:54.

In one specific embodiment, the T cell activating bispecific antigenbinding molecule does not induce killing of a normal cells having lessthan about 1000 copies of FolR1 its surface.

In addition to the above advantageous characteristics, one embodiment ofthe invention does not require chemical cross linking or a hybridapproach to be produced. Accordingly, in one embodiment, the inventionprovides for T cell activating bispecific antigen binding moleculecapable of production in CHO cells. In one embodiment, the T cellactivating bispecific antigen binding molecule comprises humanized andhuman polypeptides. In one embodiment, the T cell activating bispecificantigen binding molecule does not cause FcgR crosslinking. In one suchembodiment, the T cell activating bispecific antigen binding molecule iscapable of production in CHO cells and comprises a CD3 antigen bindingmoiety that comprises the heavy chain CDR1 of SEQ ID NO: 37, the heavychain CDR2 of SEQ ID NO: 38, the heavy chain CDR3 of SEQ ID NO:39, thelight chain CDR1 of SEQ ID NO: 32, the light chain CDR2 of SEQ ID NO:33, and the light chain CDR3 of SEQ ID NO:34 and two FolR1 antigenbinding moieties that each comprise the heavy chain CDR1 of SEQ ID NO:8, the heavy chain CDR2 of SEQ ID NO: 9, the heavy chain CDR3 of SEQ IDNO:50, the light chain CDR1 of SEQ ID NO: 52, the light chain CDR2 ofSEQ ID NO: 53, and the light chain CDR3 of SEQ ID NO:54.

As noted above, some embodiments contemplated herein include T cellactivating bispecific antigen binding molecules having two bindingmoieties that confer specific binding to FolR1 and one binding moietythat confers specificity to the T cell activating antigen CD3, whereineach individual FolR1 binding moiety engages the antigen with lowaffinity. Because the molecule comprises two antigen binding moietiesthat confer binding to FolR1, the overall avidity of the molecule,nevertheless, provides effective binding to FolR1-expressing targetcells and activation of T cells to induce T cell effector function.Considering that while FolR1 is expressed at various level on tumorcells, it is also expressed at very low levels (e.g., less than about1000 copies on the cell surface) in certain normal cells, one of skillin the art can readily recognize the advantageous efficiency of such amolecule for use as a therapeutic agent. Such molecule selectivelytargets tumor cells over normal cells. Such molecule, thus, can beadministered to an individual in need thereof with significantly lessconcern about toxicity resulting from FolR1 positive normal cellscompared to molecules that bind to FolR1 with high affinity to induceeffector function. In a preferred embodiment, the T cell activatingbispecific antigen binding molecules have a monovalent binding affinityto huFolR1 in the micromolar range and an avidity to huFolR1 in thenanomolar range.

In one embodiment, the T cell activating bispecific antigen bindingmolecule binds human FolR1 with an apparent K_(D) of about 10 nM toabout 40 nM. In one embodiment, the T cell activating bispecific antigenbinding molecule binds human FolR1 with an apparent K_(D) of about 10nM. In one embodiment, the T cell activating bispecific antigen bindingmolecule binds human and cynomolgus FolR1 with an apparent K_(D) ofabout 10 nM and about 30 nM, respectively. In one embodiment, the T cellactivating bispecific antigen binding molecule binds human FolR1 with amonovalent binding K_(D) of at least about 1000 nM. In one embodiment,the T cell activating bispecific antigen binding molecule binds humanFolR1 with a monovalent binding K_(D) of about 1400 nM. In oneembodiment, the T cell activating bispecific antigen binding moleculebinds human FolR1 with a monovalent binding K_(D) of about 1400 nM andto cynomolgus FolR1 with a monovalent binding K_(D) of about 5600 nM. Inone embodiment, the T cell activating bispecific antigen bindingmolecule binds human FolR1 with an apparent K_(D) of about 10 nM andwith a monovalent binding K_(D) of about 1400 nM.

In one embodiment, the T cell activating bispecific antigen bindingmolecule binds human FolR1 with an apparent K_(D) of about 5.36 pM toabout 4 nM. In one embodiment, the T cell activating bispecific antigenbinding molecule binds human and cynomolgus FolR1 with an apparent K_(D)of about 4 nM. In one embodiment, the T cell activating bispecificantigen binding molecule binds murine FolR1 with an apparent K_(D) ofabout 1.5 nM. In one embodiment, the T cell activating bispecificantigen binding molecule binds human FolR1 with a monovalent bindingK_(D) of at least about 1000 nM. In a specific embodiment, the T cellactivating bispecific antigen binding molecule binds human andcynomolgus FolR1 with an apparent K_(D) of about 4 nM, binds murineFolR1 with an apparent K_(D) of about 1.5 nM, and comprises a CD3antigen binding moiety that comprises the heavy chain CDR1 of SEQ ID NO:37, the heavy chain CDR2 of SEQ ID NO: 38, the heavy chain CDR3 of SEQID NO:39, the light chain CDR1 of SEQ ID NO: 32, the light chain CDR2 ofSEQ ID NO: 33, and the light chain CDR3 of SEQ ID NO:34 and two FolR1antigen binding moieties that each comprise the heavy chain CDR1 of SEQID NO: 8, the heavy chain CDR2 of SEQ ID NO: 9, the heavy chain CDR3 ofSEQ ID NO:50, the light chain CDR1 of SEQ ID NO: 52, the light chainCDR2 of SEQ ID NO: 53, and the light chain CDR3 of SEQ ID NO:54. In oneembodiment, the T cell activating bispecific antigen binding moleculebinds human FolR1 with a monovalent binding K_(D) of at least about 1000nM and comprises a CD3 antigen binding moiety that comprises the heavychain CDR1 of SEQ ID NO: 37, the heavy chain CDR2 of SEQ ID NO: 38, theheavy chain CDR3 of SEQ ID NO:39, the light chain CDR1 of SEQ ID NO: 32,the light chain CDR2 of SEQ ID NO: 33, and the light chain CDR3 of SEQID NO:34 and two FolR1 antigen binding moieties that each comprise theheavy chain CDR1 of SEQ ID NO: 8, the heavy chain CDR2 of SEQ ID NO: 9,the heavy chain CDR3 of SEQ ID NO:50, the light chain CDR1 of SEQ ID NO:52, the light chain CDR2 of SEQ ID NO: 53, and the light chain CDR3 ofSEQ ID NO:54.

As described above, the T cell activating bispecific antigen bindingmolecules contemplated herein can induce T cell effector function, e.g.,cell surface marker expression, cytokine production, T cell mediatedkilling. In one embodiment, the T cell activating bispecific antigenbinding molecule induces T cell mediated killing of the FolR1-expressingtarget cell, such as a human tumor cell, in vitro. In one embodiment,the T cell is a CD8 positive T cell. Examples of FolR1-expressing humantumor cells include but are not limited to Hela, Skov-3, HT-29, andHRCEpiC cells. Other FolR1 positive human cancer cells that can be usedfor in vitro testing are readily available to the skilled artisan. Inone embodiment, the T cell activating bispecific antigen bindingmolecule induces T cell mediated killing of the FolR1-expressing humantumor cell in vitro with an EC50 of between about 36 pM and about 39573pM after 24 hours. Specifically contemplated are T cell activatingbispecific antigen binding molecules that induce T cell mediated killingof the FolR1-expressing tumor cell in vitro with an EC50 of about 36 pMafter 24 hours. In one embodiment, the T cell activating bispecificantigen binding molecule induces T cell mediated killing of theFolR1-expressing tumor cell in vitro with an EC50 of about 178.4 pMafter 24 hours. In one embodiment, the T cell activating bispecificantigen binding molecule induces T cell mediated killing of theFolR1-expressing tumor cell in vitro with an EC50 of about 134.5 pM orgreater after 48 hours. The EC50 can be measure by methods known in theart, for example by methods disclosed herein by the examples.

In one embodiment, the T cell activating bispecific antigen bindingmolecule of any of the above embodiments induces upregulation of cellsurface expression of at least one of CD25 and CD69 on the T cell asmeasured by flow cytometry. In one embodiment, the T cell is a CD4positive T cell or a CD8 positive T cell.

In one embodiment, the T cell activating bispecific antigen bindingmolecule of any of the above embodiments binds to FolR1 expressed on ahuman tumor cell. In one embodiment, the T cell activating bispecificantigen binding molecule of any of the above embodiments binds to aconformational epitope on human FolR1. In one embodiment, the T cellactivating bispecific antigen binding molecule of any of the aboveembodiments does not bind to human Folate Receptor 2 (FolR2) or to humanFolate Receptor 3 (FolR3). In one embodiment of the T cell activatingbispecific antigen binding molecule of any of the above embodiments, theantigen binding moiety binds to a FolR1 polypeptide comprising the aminoacids 25 to 234 of human FolR1 (SEQ ID NO:227). In one embodiment of theT cell activating bispecific antigen binding molecule of any of theabove embodiments, the FolR1 antigen binding moiety binds to a FolR1polypeptide comprising the amino acid sequence of SEQ ID NO:227, to aFolR1 polypeptide comprising the amino acid sequence of SEQ ID NO:230and to a FolR1 polypeptide comprising the amino acid sequence of SEQ IDNO:231, and wherein the FolR1 antigen binding moiety does not bind to aFolR polypeptide comprising the amino acid sequence of SEQ ID NOs:228 or229. In one specific embodiment, the T cell activating bispecificantigen binding molecule comprises a FolR1 antigen binding moiety thatbinds to a FolR1 polypeptide comprising the amino acid sequence of SEQID NOs:227, 230 and 231, and wherein the FolR1 antigen binding moietydoes not bind to a FolR polypeptide comprising the amino acid sequenceof SEQ ID NOs:228 or 229, and comprises a CD3 antigen binding moietythat comprises the heavy chain CDR1 of SEQ ID NO: 37, the heavy chainCDR2 of SEQ ID NO: 38, the heavy chain CDR3 of SEQ ID NO:39, the lightchain CDR1 of SEQ ID NO: 32, the light chain CDR2 of SEQ ID NO: 33, andthe light chain CDR3 of SEQ ID NO:34 and two FolR1 antigen bindingmoieties that each comprise the heavy chain CDR1 of SEQ ID NO: 8, theheavy chain CDR2 of SEQ ID NO: 9, the heavy chain CDR3 of SEQ ID NO:50,the light chain CDR1 of SEQ ID NO: 52, the light chain CDR2 of SEQ IDNO: 53, and the light chain CDR3 of SEQ ID NO:54.

In one embodiment of the T cell activating bispecific antigen bindingmolecule of any of the above embodiments, the FolR1 antigen bindingmoiety binds to a FolR1 polypeptide comprising the amino acid sequenceof SEQ ID NO:227 and to a FolR1 polypeptide comprising the amino acidsequence of SEQ ID NO:231, and wherein the FolR1 antigen binding moietydoes not bind to a FolR polypeptide comprising the amino acid sequenceof SEQ ID NOs:228, 229 or 230. In one specific embodiment, the T cellactivating bispecific antigen binding molecule comprises a FolR1 antigenbinding moiety that binds to a FolR1 polypeptide comprising the aminoacid sequence of SEQ ID NO:227 and to a FolR1 polypeptide comprising theamino acid sequence of SEQ ID NO:231, and wherein the FolR1 antigenbinding moiety does not bind to a FolR polypeptide comprising the aminoacid sequence of SEQ ID NOs:228, 229 or 230, and comprises a CD3 antigenbinding moiety that comprises the heavy chain CDR1 of SEQ ID NO: 37, theheavy chain CDR2 of SEQ ID NO: 38, the heavy chain CDR3 of SEQ ID NO:39,the light chain CDR1 of SEQ ID NO: 32, the light chain CDR2 of SEQ IDNO: 33, and the light chain CDR3 of SEQ ID NO:34 and two FolR1 antigenbinding moieties that each comprise the heavy chain CDR1 of SEQ ID NO:16, the heavy chain CDR2 of SEQ ID NO: 275, the heavy chain CDR3 of SEQID NO:315, the light chain CDR1 of SEQ ID NO: 32, the light chain CDR2of SEQ ID NO: 33, and the light chain CDR3 of SEQ ID NO:34.

With respect to the FolR1, the T cell activating bispecific antigenbinding molecules contemplated herein can have agonist, antagonist orneutral effect. Examples of agonist effect include induction orenhancement of signaling through the FolR1 upon engagement by the FolR1binding moiety with the FolR1 receptor on the target cell. Examples ofantagonist activity include abrogation or reduction of signaling throughthe FolR1 upon engagement by the FolR1 binding moiety with the FolR1receptor on the target cell. This can, for example, occur by blocking orreducing the interaction between folate with FolR1. Sequence variants ofthe embodiments disclosed herein having lower affinity while retainingthe above described biological properties are specifically contemplated.

Immunoconjugates

The invention also pertains to immunoconjugates comprising a T cellactivating bispecific antigen binding molecule conjugated to a cytotoxicagent such as a chemotherapeutic agent, a growth inhibitory agent, atoxin (e.g., an enzymatically active toxin of bacterial, fungal, plant,or animal origin, or fragments thereof), or a radioactive isotope (i.e.,a radioconjugate).

Polynucleotides

The invention further provides isolated polynucleotides encoding a Tcell activating bispecific antigen binding molecule as described hereinor a fragment thereof.

Polynucleotides of the invention include those that are at least about80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to thesequences set forth in SEQ ID NOs:151-226 including functional fragmentsor variants thereof.

The polynucleotides encoding T cell activating bispecific antigenbinding molecules of the invention may be expressed as a singlepolynucleotide that encodes the entire T cell activating bispecificantigen binding molecule or as multiple (e.g., two or more)polynucleotides that are co-expressed. Polypeptides encoded bypolynucleotides that are co-expressed may associate through, e.g.,disulfide bonds or other means to form a functional T cell activatingbispecific antigen binding molecule. For example, the light chainportion of an antigen binding moiety may be encoded by a separatepolynucleotide from the portion of the T cell activating bispecificantigen binding molecule comprising the heavy chain portion of theantigen binding moiety, an Fc domain subunit and optionally (part of)another antigen binding moiety. When co-expressed, the heavy chainpolypeptides will associate with the light chain polypeptides to formthe antigen binding moiety. In another example, the portion of the Tcell activating bispecific antigen binding molecule comprising one ofthe two Fc domain subunits and optionally (part of) one or more antigenbinding moieties could be encoded by a separate polynucleotide from theportion of the T cell activating bispecific antigen binding moleculecomprising the the other of the two Fc domain subunits and optionally(part of) an antigen binding moiety. When co-expressed, the Fc domainsubunits will associate to form the Fc domain.

In some embodiments, the isolated polynucleotide encodes the entire Tcell activating bispecific antigen binding molecule according to theinvention as described herein. In other embodiments, the isolatedpolynucleotide encodes a polypeptides comprised in the T cell activatingbispecific antigen binding molecule according to the invention asdescribed herein.

In another embodiment, the present invention is directed to an isolatedpolynucleotide encoding a T cell activating bispecific antigen bindingmolecule of the invention or a fragment thereof, wherein thepolynucleotide comprises a sequence that encodes a variable regionsequence as shown in SEQ ID NOs 151, 152, 153, 154, 155, 156, 157, 158,159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172,173, 174, 175, 176, 177, 178, 179, 180, 181, 182 and 205, 206, 207, 208,209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222,223. In another embodiment, the present invention is directed to anisolated polynucleotide encoding a T cell activating bispecific antigenbinding molecule or fragment thereof, wherein the polynucleotidecomprises a sequence that encodes a polypeptide sequence as shown in SEQID NOs:1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36,37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54,55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 1, 72,73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90,91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106,107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120,121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134,135, 136, 137, 138, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236,237, 238, 239, 240, 241, 242, 243, 244.

In another embodiment, the invention is further directed to an isolatedpolynucleotide encoding a T cell activating bispecific antigen bindingmolecule of the invention or a fragment thereof, wherein thepolynucleotide comprises a sequence that is at least about 80%, 85%,90%, 95%, 96%, 97%, 98%, or 99% identical to a nucleotide sequence shownin SEQ ID NOs 97, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161,162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175,176, 177, 178, 179, 180, 181, 182, 183, 184, 189, 190, 191, 192, 193,194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207,208, 209, 210, 211, 12, 213, 214, 215, 216, 217, 218, 219, 220, 221,222, 223, 224, 225, 226, 246, 247. In another embodiment, the inventionis directed to an isolated polynucleotide encoding a T cell activatingbispecific antigen binding molecule of the invention or a fragmentthereof, wherein the polynucleotide comprises a nucleic acid sequenceshown in SEQ ID NOs 97, 151, 152, 153, 154, 155, 156, 157, 158, 159,160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173,174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 189, 190, 191,192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205,206, 207, 208, 209, 210, 211, 12, 213, 214, 215, 216, 217, 218, 219,220, 221, 222, 223, 224, 225, 226, 246, 247.

In another embodiment, the invention is directed to an isolatedpolynucleotide encoding a T cell activating bispecific antigen bindingmolecule of the invention or a fragment thereof, wherein thepolynucleotide comprises a sequence that encodes a variable regionsequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or99% identical to an amino acid sequence in SEQ ID NOs 1, 2, 3, 4, 5, 6,7, 11, 13, 15, 19, 21, 12, 25, 27, 29, 31, 36, 41, 45, 49, 51, 55, 58,62, 64, 66, 68, 70, 72, 74, 76, 78, 82, 113, 114, 115, 116, 117, 118,119, 12, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132,133, 134, 135. In another embodiment, the invention is directed to anisolated polynucleotide encoding a T cell activating bispecific antigenbinding molecule or fragment thereof, wherein the polynucleotidecomprises a sequence that encodes a polypeptide comprising one or moresequences that are at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%identical to an amino acid sequence in SEQ ID NOs: 8, 9, 50, 37, 38, and39. The invention encompasses an isolated polynucleotide encoding a Tcell activating bispecific antigen binding molecule of the invention ora fragment thereof, wherein the polynucleotide comprises a sequence thatencodes the variable region sequence of SEQ ID NOs 151, 152, 153, 154,155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168,169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182 and205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218,219, 220, 221, 222, 223 with conservative amino acid substitutions. Theinvention also encompasses an isolated polynucleotide encoding a T cellactivating bispecific antigen binding molecule of the invention orfragment thereof, wherein the polynucleotide comprises a sequence thatencodes the polypeptide sequence of SEQ ID NOs 1, 2, 3, 4, 5, 6, 7, 8,9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26,27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44,45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62,63, 64, 65, 66, 67, 68, 69, 70, 1, 72, 73, 74, 75, 76, 77, 78, 79, 80,81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98,99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112,113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126,127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138 and 227, 228,229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242,243, 244 with conservative amino acid substitutions.

In certain embodiments the polynucleotide or nucleic acid is DNA. Inother embodiments, a polynucleotide of the present invention is RNA, forexample, in the form of messenger RNA (mRNA). RNA of the presentinvention may be single stranded or double stranded.

Recombinant Methods

T cell activating bispecific antigen binding molecules of the inventionmay be obtained, for example, by solid-state peptide synthesis (e.g.Merrifield solid phase synthesis) or recombinant production. Forrecombinant production one or more polynucleotide encoding the T cellactivating bispecific antigen binding molecule (fragment), e.g., asdescribed above, is isolated and inserted into one or more vectors forfurther cloning and/or expression in a host cell. Such polynucleotidemay be readily isolated and sequenced using conventional procedures. Inone embodiment a vector, preferably an expression vector, comprising oneor more of the polynucleotides of the invention is provided. Methodswhich are well known to those skilled in the art can be used toconstruct expression vectors containing the coding sequence of a T cellactivating bispecific antigen binding molecule (fragment) along withappropriate transcriptional/translational control signals. These methodsinclude in vitro recombinant DNA techniques, synthetic techniques and invivo recombination/genetic recombination. See, for example, thetechniques described in Maniatis et al., MOLECULAR CLONING: A LABORATORYMANUAL, Cold Spring Harbor Laboratory, N.Y. (1989); and Ausubel et al.,CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, Greene Publishing Associates andWiley Interscience, N.Y. (1989). The expression vector can be part of aplasmid, virus, or may be a nucleic acid fragment. The expression vectorincludes an expression cassette into which the polynucleotide encodingthe T cell activating bispecific antigen binding molecule (fragment)(i.e. the coding region) is cloned in operable association with apromoter and/or other transcription or translation control elements. Asused herein, a “coding region” is a portion of nucleic acid whichconsists of codons translated into amino acids. Although a “stop codon”(TAG, TGA, or TAA) is not translated into an amino acid, it may beconsidered to be part of a coding region, if present, but any flankingsequences, for example promoters, ribosome binding sites,transcriptional terminators, introns, 5′ and 3′ untranslated regions,and the like, are not part of a coding region. Two or more codingregions can be present in a single polynucleotide construct, e.g. on asingle vector, or in separate polynucleotide constructs, e.g. onseparate (different) vectors. Furthermore, any vector may contain asingle coding region, or may comprise two or more coding regions, e.g. avector of the present invention may encode one or more polypeptides,which are post- or co-translationally separated into the final proteinsvia proteolytic cleavage. In addition, a vector, polynucleotide, ornucleic acid of the invention may encode heterologous coding regions,either fused or unfused to a polynucleotide encoding the T cellactivating bispecific antigen binding molecule (fragment) of theinvention, or variant or derivative thereof. Heterologous coding regionsinclude without limitation specialized elements or motifs, such as asecretory signal peptide or a heterologous functional domain. Anoperable association is when a coding region for a gene product, e.g. apolypeptide, is associated with one or more regulatory sequences in sucha way as to place expression of the gene product under the influence orcontrol of the regulatory sequence(s). Two DNA fragments (such as apolypeptide coding region and a promoter associated therewith) are“operably associated” if induction of promoter function results in thetranscription of mRNA encoding the desired gene product and if thenature of the linkage between the two DNA fragments does not interferewith the ability of the expression regulatory sequences to direct theexpression of the gene product or interfere with the ability of the DNAtemplate to be transcribed. Thus, a promoter region would be operablyassociated with a nucleic acid encoding a polypeptide if the promoterwas capable of effecting transcription of that nucleic acid. Thepromoter may be a cell-specific promoter that directs substantialtranscription of the DNA only in predetermined cells. Othertranscription control elements, besides a promoter, for exampleenhancers, operators, repressors, and transcription termination signals,can be operably associated with the polynucleotide to directcell-specific transcription. Suitable promoters and other transcriptioncontrol regions are disclosed herein. A variety of transcription controlregions are known to those skilled in the art. These include, withoutlimitation, transcription control regions, which function in vertebratecells, such as, but not limited to, promoter and enhancer segments fromcytomegaloviruses (e.g. the immediate early promoter, in conjunctionwith intron-A), simian virus 40 (e.g. the early promoter), andretroviruses (such as, e.g. Rous sarcoma virus). Other transcriptioncontrol regions include those derived from vertebrate genes such asactin, heat shock protein, bovine growth hormone and rabbit â-globin, aswell as other sequences capable of controlling gene expression ineukaryotic cells. Additional suitable transcription control regionsinclude tissue-specific promoters and enhancers as well as induciblepromoters (e.g. promoters inducible tetracyclins). Similarly, a varietyof translation control elements are known to those of ordinary skill inthe art. These include, but are not limited to ribosome binding sites,translation initiation and termination codons, and elements derived fromviral systems (particularly an internal ribosome entry site, or IRES,also referred to as a CITE sequence). The expression cassette may alsoinclude other features such as an origin of replication, and/orchromosome integration elements such as retroviral long terminal repeats(LTRs), or adeno-associated viral (AAV) inverted terminal repeats(ITRs).

Polynucleotide and nucleic acid coding regions of the present inventionmay be associated with additional coding regions which encode secretoryor signal peptides, which direct the secretion of a polypeptide encodedby a polynucleotide of the present invention. For example, if secretionof the T cell activating bispecific antigen binding molecule is desired,DNA encoding a signal sequence may be placed upstream of the nucleicacid encoding a T cell activating bispecific antigen binding molecule ofthe invention or a fragment thereof. According to the signal hypothesis,proteins secreted by mammalian cells have a signal peptide or secretoryleader sequence which is cleaved from the mature protein once export ofthe growing protein chain across the rough endoplasmic reticulum hasbeen initiated. Those of ordinary skill in the art are aware thatpolypeptides secreted by vertebrate cells generally have a signalpeptide fused to the N-terminus of the polypeptide, which is cleavedfrom the translated polypeptide to produce a secreted or “mature” formof the polypeptide. In certain embodiments, the native signal peptide,e.g. an immunoglobulin heavy chain or light chain signal peptide isused, or a functional derivative of that sequence that retains theability to direct the secretion of the polypeptide that is operablyassociated with it. Alternatively, a heterologous mammalian signalpeptide, or a functional derivative thereof, may be used. For example,the wild-type leader sequence may be substituted with the leadersequence of human tissue plasminogen activator (TPA) or mouseβ-glucuronidase.

DNA encoding a short protein sequence that could be used to facilitatelater purification (e.g. a histidine tag) or assist in labeling the Tcell activating bispecific antigen binding molecule may be includedwithin or at the ends of the T cell activating bispecific antigenbinding molecule (fragment) encoding polynucleotide.

In a further embodiment, a host cell comprising one or morepolynucleotides of the invention is provided. In certain embodiments ahost cell comprising one or more vectors of the invention is provided.The polynucleotides and vectors may incorporate any of the features,singly or in combination, described herein in relation topolynucleotides and vectors, respectively. In one such embodiment a hostcell comprises (e.g. has been transformed or transfected with) a vectorcomprising a polynucleotide that encodes (part of) a T cell activatingbispecific antigen binding molecule of the invention. As used herein,the term “host cell” refers to any kind of cellular system which can beengineered to generate the T cell activating bispecific antigen bindingmolecules of the invention or fragments thereof. Host cells suitable forreplicating and for supporting expression of T cell activatingbispecific antigen binding molecules are well known in the art. Suchcells may be transfected or transduced as appropriate with theparticular expression vector and large quantities of vector containingcells can be grown for seeding large scale fermenters to obtainsufficient quantities of the T cell activating bispecific antigenbinding molecule for clinical applications. Suitable host cells includeprokaryotic microorganisms, such as E. coli, or various eukaryoticcells, such as Chinese hamster ovary cells (CHO), insect cells, or thelike. For example, polypeptides may be produced in bacteria inparticular when glycosylation is not needed. After expression, thepolypeptide may be isolated from the bacterial cell paste in a solublefraction and can be further purified. In addition to prokaryotes,eukaryotic microbes such as filamentous fungi or yeast are suitablecloning or expression hosts for polypeptide-encoding vectors, includingfungi and yeast strains whose glycosylation pathways have been“humanized”, resulting in the production of a polypeptide with apartially or fully human glycosylation pattern. See Gerngross, NatBiotech 22, 1409-1414 (2004), and Li et al., Nat Biotech 24, 210-215(2006). Suitable host cells for the expression of (glycosylated)polypeptides are also derived from multicellular organisms(invertebrates and vertebrates). Examples of invertebrate cells includeplant and insect cells.

Numerous baculoviral strains have been identified which may be used inconjunction with insect cells, particularly for transfection ofSpodoptera frugiperda cells. Plant cell cultures can also be utilized ashosts. See e.g. U.S. Pat. Nos. 5,959,177, 6,040,498, 6,420,548,7,125,978, and 6,417,429 (describing PLANTIBODIES™ technology forproducing antibodies in transgenic plants). Vertebrate cells may also beused as hosts. For example, mammalian cell lines that are adapted togrow in suspension may be useful. Other examples of useful mammalianhost cell lines are monkey kidney CV1 line transformed by SV40 (COS-7);human embryonic kidney line (293 or 293T cells as described, e.g., inGraham et al., J Gen Virol 36, 59 (1977)), baby hamster kidney cells(BHK), mouse sertoli cells (TM4 cells as described, e.g., in Mather,Biol Reprod 23, 243-251 (1980)), monkey kidney cells (CV1), Africangreen monkey kidney cells (VERO-76), human cervical carcinoma cells(HELA), canine kidney cells (MDCK), buffalo rat liver cells (BRL 3A),human lung cells (W138), human liver cells (Hep G2), mouse mammary tumorcells (MMT 060562), TRI cells (as described, e.g., in Mather et al.,Annals N.Y. Acad Sci 383, 44-68 (1982)), MRC 5 cells, and FS4 cells.Other useful mammalian host cell lines include Chinese hamster ovary(CHO) cells, including dhfr⁻ CHO cells (Urlaub et al., Proc Natl AcadSci USA 77, 4216 (1980)); and myeloma cell lines such as YO, NS0, P3X63and Sp2/0. For a review of certain mammalian host cell lines suitablefor protein production, see, e.g., Yazaki and Wu, Methods in MolecularBiology, Vol. 248 (B. K. C. Lo, ed., Humana Press, Totowa, N.J.), pp.255-268 (2003). Host cells include cultured cells, e.g., mammaliancultured cells, yeast cells, insect cells, bacterial cells and plantcells, to name only a few, but also cells comprised within a transgenicanimal, transgenic plant or cultured plant or animal tissue. In oneembodiment, the host cell is a eukaryotic cell, preferably a mammaliancell, such as a Chinese Hamster Ovary (CHO) cell, a human embryonickidney (HEK) cell or a lymphoid cell (e.g., Y0, NS0, Sp20 cell).

Standard technologies are known in the art to express foreign genes inthese systems. Cells expressing a polypeptide comprising either theheavy or the light chain of an antigen binding domain such as anantibody, may be engineered so as to also express the other of theantibody chains such that the expressed product is an antibody that hasboth a heavy and a light chain.

In one embodiment, a method of producing a T cell activating bispecificantigen binding molecule according to the invention is provided, whereinthe method comprises culturing a host cell comprising a polynucleotideencoding the T cell activating bispecific antigen binding molecule, asprovided herein, under conditions suitable for expression of the T cellactivating bispecific antigen binding molecule, and recovering the Tcell activating bispecific antigen binding molecule from the host cell(or host cell culture medium).

The components of the T cell activating bispecific antigen bindingmolecule are genetically fused to each other. T cell activatingbispecific antigen binding molecule can be designed such that itscomponents are fused directly to each other or indirectly through alinker sequence. The composition and length of the linker may bedetermined in accordance with methods well known in the art and may betested for efficacy. Examples of linker sequences between differentcomponents of T cell activating bispecific antigen binding molecules arefound in the sequences provided herein. Additional sequences may also beincluded to incorporate a cleavage site to separate the individualcomponents of the fusion if desired, for example an endopeptidaserecognition sequence.

In certain embodiments the one or more antigen binding moieties of the Tcell activating bispecific antigen binding molecules comprise at leastan antibody variable region capable of binding an antigenic determinant.Variable regions can form part of and be derived from naturally ornon-naturally occurring antibodies and fragments thereof. Methods toproduce polyclonal antibodies and monoclonal antibodies are well knownin the art (see e.g. Harlow and Lane, “Antibodies, a laboratory manual”,Cold Spring Harbor Laboratory, 1988). Non-naturally occurring antibodiescan be constructed using solid phase-peptide synthesis, can be producedrecombinantly (e.g. as described in U.S. Pat. No. 4,186,567) or can beobtained, for example, by screening combinatorial libraries comprisingvariable heavy chains and variable light chains (see e.g. U.S. Pat. No.5,969,108, McCafferty).

Any animal species of antibody, antibody fragment, antigen bindingdomain or variable region can be used in the T cell activatingbispecific antigen binding molecules of the invention. Non-limitingantibodies, antibody fragments, antigen binding domains or variableregions useful in the present invention can be of murine, primate, orhuman origin. If the T cell activating bispecific antigen bindingmolecule is intended for human use, a chimeric form of antibody may beused wherein the constant regions of the antibody are from a human. Ahumanized or fully human form of the antibody can also be prepared inaccordance with methods well known in the art (see e. g. U.S. Pat. No.5,565,332 to Winter). Humanization may be achieved by various methodsincluding, but not limited to (a) grafting the non-human (e.g., donorantibody) CDRs onto human (e.g. recipient antibody) framework andconstant regions with or without retention of critical frameworkresidues (e.g. those that are important for retaining good antigenbinding affinity or antibody functions), (b) grafting only the non-humanspecificity-determining regions (SDRs or a-CDRs; the residues criticalfor the antibody-antigen interaction) onto human framework and constantregions, or (c) transplanting the entire non-human variable domains, but“cloaking” them with a human-like section by replacement of surfaceresidues. Humanized antibodies and methods of making them are reviewed,e.g., in Almagro and Fransson, Front Biosci 13, 1619-1633 (2008), andare further described, e.g., in Riechmann et al., Nature 332, 323-329(1988); Queen et al., Proc Natl Acad Sci USA 86, 10029-10033 (1989);U.S. Pat. Nos. 5,821,337, 7,527,791, 6,982,321, and 7,087,409; Jones etal., Nature 321, 522-525 (1986); Morrison et al., Proc Natl Acad Sci 81,6851-6855 (1984); Morrison and Oi, Adv Immunol 44, 65-92 (1988);Verhoeyen et al., Science 239, 1534-1536 (1988); Padlan, Molec Immun31(3), 169-217 (1994); Kashmiri et al., Methods 36, 25-34 (2005)(describing SDR (a-CDR) grafting); Padlan, Mol Immunol 28, 489-498(1991) (describing “resurfacing”); Dall'Acqua et al., Methods 36, 43-60(2005) (describing “FR shuffling”); and Osbourn et al., Methods 36,61-68 (2005) and Klimka et al., Br J Cancer 83, 252-260 (2000)(describing the “guided selection” approach to FR shuffling). Humanantibodies and human variable regions can be produced using varioustechniques known in the art. Human antibodies are described generally invan Dijk and van de Winkel, Curr Opin Pharmacol 5, 368-74 (2001) andLonberg, Curr Opin Immunol 20, 450-459 (2008). Human variable regionscan form part of and be derived from human monoclonal antibodies made bythe hybridoma method (see e.g. Monoclonal Antibody Production Techniquesand Applications, pp. 51-63 (Marcel Dekker, Inc., New York, 1987)).Human antibodies and human variable regions may also be prepared byadministering an immunogen to a transgenic animal that has been modifiedto produce intact human antibodies or intact antibodies with humanvariable regions in response to antigenic challenge (see e.g. Lonberg,Nat Biotech 23, 1117-1125 (2005). Human antibodies and human variableregions may also be generated by isolating Fv clone variable regionsequences selected from human-derived phage display libraries (see e.g.,Hoogenboom et al. in Methods in Molecular Biology 178, 1-37 (O'Brien etal., ed., Human Press, Totowa, N.J., 2001); and McCafferty et al.,Nature 348, 552-554; Clackson et al., Nature 352, 624-628 (1991)). Phagetypically display antibody fragments, either as single-chain Fv (scFv)fragments or as Fab fragments.

In certain embodiments, the antigen binding moieties useful in thepresent invention are engineered to have enhanced binding affinityaccording to, for example, the methods disclosed in U.S. Pat. Appl.Publ. No. 2004/0132066, the entire contents of which are herebyincorporated by reference. The ability of the T cell activatingbispecific antigen binding molecule of the invention to bind to aspecific antigenic determinant can be measured either through anenzyme-linked immunosorbent assay (ELISA) or other techniques familiarto one of skill in the art, e.g. surface plasmon resonance technique(analyzed on a BIACORE T100 system) (Liljeblad, et al., Glyco J 17,323-329 (2000)), and traditional binding assays (Heeley, Endocr Res 28,217-229 (2002)). Competition assays may be used to identify an antibody,antibody fragment, antigen binding domain or variable domain thatcompetes with a reference antibody for binding to a particular antigen,e.g. an antibody that competes with the V9 antibody for binding to CD3.In certain embodiments, such a competing antibody binds to the sameepitope (e.g. a linear or a conformational epitope) that is bound by thereference antibody. Detailed exemplary methods for mapping an epitope towhich an antibody binds are provided in Morris (1996) “Epitope MappingProtocols,” in Methods in Molecular Biology vol. 66 (Humana Press,Totowa, N.J.). In an exemplary competition assay, immobilized antigen(e.g. CD3) is incubated in a solution comprising a first labeledantibody that binds to the antigen (e.g. V9 antibody, described in U.S.Pat. No. 6,054,297) and a second unlabeled antibody that is being testedfor its ability to compete with the first antibody for binding to theantigen. The second antibody may be present in a hybridoma supernatant.As a control, immobilized antigen is incubated in a solution comprisingthe first labeled antibody but not the second unlabeled antibody. Afterincubation under conditions permissive for binding of the first antibodyto the antigen, excess unbound antibody is removed, and the amount oflabel associated with immobilized antigen is measured. If the amount oflabel associated with immobilized antigen is substantially reduced inthe test sample relative to the control sample, then that indicates thatthe second antibody is competing with the first antibody for binding tothe antigen. See Harlow and Lane (1988) Antibodies: A Laboratory Manualch.14 (Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.).

T cell activating bispecific antigen binding molecules prepared asdescribed herein may be purified by art-known techniques such as highperformance liquid chromatography, ion exchange chromatography, gelelectrophoresis, affinity chromatography, size exclusion chromatography,and the like. The actual conditions used to purify a particular proteinwill depend, in part, on factors such as net charge, hydrophobicity,hydrophilicity etc., and will be apparent to those having skill in theart. For affinity chromatography purification an antibody, ligand,receptor or antigen can be used to which the T cell activatingbispecific antigen binding molecule binds. For example, for affinitychromatography purification of T cell activating bispecific antigenbinding molecules of the invention, a matrix with protein A or protein Gmay be used. Sequential Protein A or G affinity chromatography and sizeexclusion chromatography can be used to isolate a T cell activatingbispecific antigen binding molecule essentially as described in theExamples. The purity of the T cell activating bispecific antigen bindingmolecule can be determined by any of a variety of well known analyticalmethods including gel electrophoresis, high pressure liquidchromatography, and the like. For example, the heavy chain fusionproteins expressed as described in the Examples were shown to be intactand properly assembled as demonstrated by reducing SDS-PAGE (see e.g.FIG. 2). Three bands were resolved at approximately Mr 25,000, Mr 50,000and Mr 75,000, corresponding to the predicted molecular weights of the Tcell activating bispecific antigen binding molecule light chain, heavychain and heavy chain/light chain fusion protein.

Assays

T cell activating bispecific antigen binding molecules provided hereinmay be identified, screened for, or characterized for theirphysical/chemical properties and/or biological activities by variousassays known in the art.

Affinity Assays

The affinity of the T cell activating bispecific antigen bindingmolecule for an Fc receptor or a target antigen can be determined inaccordance with the methods set forth in the Examples by surface plasmonresonance (SPR), using standard instrumentation such as a BIAcoreinstrument (GE Healthcare), and receptors or target proteins such as maybe obtained by recombinant expression. Alternatively, binding of T cellactivating bispecific antigen binding molecules for different receptorsor target antigens may be evaluated using cell lines expressing theparticular receptor or target antigen, for example by flow cytometry(FACS). A specific illustrative and exemplary embodiment for measuringbinding affinity is described in the following and in the Examplesbelow. According to one embodiment, K_(D) is measured by surface plasmonresonance using a BIACORE® T100 machine (GE Healthcare) at 25° C.

To analyze the interaction between the Fc-portion and Fc receptors,His-tagged recombinant Fc-receptor is captured by an anti-Penta Hisantibody (“Penta His” disclosed as SEQ ID NO: 306) (Qiagen) immobilizedon CM5 chips and the bispecific constructs are used as analytes.Briefly, carboxymethylated dextran biosensor chips (CM5, GE Healthcare)are activated with N-ethyl-N′-(3-dimethylaminopropyl)-carbodiimidehydrochloride (EDC) and N-hydroxysuccinimide (NETS) according to thesupplier's instructions. Anti Penta-His antibody (“Penta-His” disclosedas SEQ ID NO: 306) is diluted with 10 mM sodium acetate, pH 5.0, to 40μg/ml before injection at a flow rate of 5 μl/min to achieveapproximately 6500 response units (RU) of coupled protein. Following theinjection of the ligand, 1 M ethanolamine is injected to block unreactedgroups. Subsequently the Fc-receptor is captured for 60 s at 4 or 10 nM.For kinetic measurements, four-fold serial dilutions of the bispecificconstruct (range between 500 nM and 4000 nM) are injected in HBS-EP (GEHealthcare, 10 mM HEPES, 150 mM NaCl, 3 mM EDTA, 0.05% Surfactant P20,pH 7.4) at 25° C. at a flow rate of 30 μl/min for 120 s.

To determine the affinity to the target antigen, bispecific constructsare captured by an anti human Fab specific antibody (GE Healthcare) thatis immobilized on an activated CM5-sensor chip surface as described forthe anti Penta-His antibody (“Penta-His” disclosed as SEQ ID NO: 306).The final amount of coupled protein is is approximately 12000 RU. Thebispecific constructs are captured for 90 s at 300 nM. The targetantigens are passed through the flow cells for 180 s at a concentrationrange from 250 to 1000 nM with a flowrate of 30 μl/min. The dissociationis monitored for 180 s.

Bulk refractive index differences are corrected for by subtracting theresponse obtained on reference flow cell. The steady state response wasused to derive the dissociation constant K_(D) by non-linear curvefitting of the Langmuir binding isotherm. Association rates (k_(on)) anddissociation rates (k_(off)) are calculated using a simple one-to-oneLangmuir binding model (BIACORE® T100 Evaluation Software version 1.1.1)by simultaneously fitting the association and dissociation sensorgrams.The equilibrium dissociation constant (K_(D)) is calculated as the ratiok_(off)/k_(on). See, e.g., Chen et al., J Mol Biol 293, 865-881 (1999).

Activity Assays

Biological activity of the T cell activating bispecific antigen bindingmolecules of the invention can be measured by various assays asdescribed in the Examples. Biological activities may for example includethe induction of proliferation of T cells, the induction of signaling inT cells, the induction of expression of activation markers in T cells,the induction of cytokine secretion by T cells, the induction of lysisof target cells such as tumor cells, and the induction of tumorregression and/or the improvement of survival.

Compositions, Formulations, and Routes of Administration

In a further aspect, the invention provides pharmaceutical compositionscomprising any of the T cell activating bispecific antigen bindingmolecules provided herein, e.g., for use in any of the below therapeuticmethods. In one embodiment, a pharmaceutical composition comprises anyof the T cell activating bispecific antigen binding molecules providedherein and a pharmaceutically acceptable carrier. In another embodiment,a pharmaceutical composition comprises any of the T cell activatingbispecific antigen binding molecules provided herein and at least oneadditional therapeutic agent, e.g., as described below.

Further provided is a method of producing a T cell activating bispecificantigen binding molecule of the invention in a form suitable foradministration in vivo, the method comprising (a) obtaining a T cellactivating bispecific antigen binding molecule according to theinvention, and (b) formulating the T cell activating bispecific antigenbinding molecule with at least one pharmaceutically acceptable carrier,whereby a preparation of T cell activating bispecific antigen bindingmolecule is formulated for administration in vivo.

Pharmaceutical compositions of the present invention comprise atherapeutically effective amount of one or more T cell activatingbispecific antigen binding molecule dissolved or dispersed in apharmaceutically acceptable carrier. The phrases “pharmaceutical orpharmacologically acceptable” refers to molecular entities andcompositions that are generally non-toxic to recipients at the dosagesand concentrations employed, i.e. do not produce an adverse, allergic orother untoward reaction when administered to an animal, such as, forexample, a human, as appropriate. The preparation of a pharmaceuticalcomposition that contains at least one T cell activating bispecificantigen binding molecule and optionally an additional active ingredientwill be known to those of skill in the art in light of the presentdisclosure, as exemplified by Remington's Pharmaceutical Sciences, 18thEd. Mack Printing Company, 1990, incorporated herein by reference.Moreover, for animal (e.g., human) administration, it will be understoodthat preparations should meet sterility, pyrogenicity, general safetyand purity standards as required by FDA Office of Biological Standardsor corresponding authorities in other countries. Preferred compositionsare lyophilized formulations or aqueous solutions. As used herein,“pharmaceutically acceptable carrier” includes any and all solvents,buffers, dispersion media, coatings, surfactants, antioxidants,preservatives (e.g. antibacterial agents, antifungal agents), isotonicagents, absorption delaying agents, salts, preservatives, antioxidants,proteins, drugs, drug stabilizers, polymers, gels, binders, excipients,disintegration agents, lubricants, sweetening agents, flavoring agents,dyes, such like materials and combinations thereof, as would be known toone of ordinary skill in the art (see, for example, Remington'sPharmaceutical Sciences, 18th Ed. Mack Printing Company, 1990, pp.1289-1329, incorporated herein by reference). Except insofar as anyconventional carrier is incompatible with the active ingredient, its usein the therapeutic or pharmaceutical compositions is contemplated.

The composition may comprise different types of carriers depending onwhether it is to be administered in solid, liquid or aerosol form, andwhether it need to be sterile for such routes of administration asinjection. T cell activating bispecific antigen binding molecules of thepresent invention (and any additional therapeutic agent) can beadministered intravenously, intradermally, intraarterially,intraperitoneally, intralesionally, intracranially, intraarticularly,intraprostatically, intrasplenically, intrarenally, intrapleurally,intratracheally, intranasally, intravitreally, intravaginally,intrarectally, intratumorally, intramuscularly, intraperitoneally,subcutaneously, subconjunctivally, intravesicularlly, mucosally,intrapericardially, intraumbilically, intraocularally, orally,topically, locally, by inhalation (e.g. aerosol inhalation), injection,infusion, continuous infusion, localized perfusion bathing target cellsdirectly, via a catheter, via a lavage, in cremes, in lipid compositions(e.g. liposomes), or by other method or any combination of the forgoingas would be known to one of ordinary skill in the art (see, for example,Remington's Pharmaceutical Sciences, 18th Ed. Mack Printing Company,1990, incorporated herein by reference). Parenteral administration, inparticular intravenous injection, is most commonly used foradministering polypeptide molecules such as the T cell activatingbispecific antigen binding molecules of the invention.

Parenteral compositions include those designed for administration byinjection, e.g. subcutaneous, intradermal, intralesional, intravenous,intraarterial intramuscular, intrathecal or intraperitoneal injection.For injection, the T cell activating bispecific antigen bindingmolecules of the invention may be formulated in aqueous solutions,preferably in physiologically compatible buffers such as Hanks'solution, Ringer's solution, or physiological saline buffer. Thesolution may contain formulatory agents such as suspending, stabilizingand/or dispersing agents. Alternatively, the T cell activatingbispecific antigen binding molecules may be in powder form forconstitution with a suitable vehicle, e.g., sterile pyrogen-free water,before use. Sterile injectable solutions are prepared by incorporatingthe T cell activating bispecific antigen binding molecules of theinvention in the required amount in the appropriate solvent with variousof the other ingredients enumerated below, as required. Sterility may bereadily accomplished, e.g., by filtration through sterile filtrationmembranes. Generally, dispersions are prepared by incorporating thevarious sterilized active ingredients into a sterile vehicle whichcontains the basic dispersion medium and/or the other ingredients. Inthe case of sterile powders for the preparation of sterile injectablesolutions, suspensions or emulsion, the preferred methods of preparationare vacuum-drying or freeze-drying techniques which yield a powder ofthe active ingredient plus any additional desired ingredient from apreviously sterile-filtered liquid medium thereof. The liquid mediumshould be suitably buffered if necessary and the liquid diluent firstrendered isotonic prior to injection with sufficient saline or glucose.The composition must be stable under the conditions of manufacture andstorage, and preserved against the contaminating action ofmicroorganisms, such as bacteria and fungi. It will be appreciated thatendotoxin contamination should be kept minimally at a safe level, forexample, less that 0.5 ng/mg protein. Suitable pharmaceuticallyacceptable carriers include, but are not limited to: buffers such asphosphate, citrate, and other organic acids; antioxidants includingascorbic acid and methionine; preservatives (such asoctadecyldimethylbenzyl ammonium chloride; hexamethonium chloride;benzalkonium chloride; benzethonium chloride; phenol, butyl or benzylalcohol; alkyl parabens such as methyl or propyl paraben; catechol;resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecularweight (less than about 10 residues) polypeptides; proteins, such asserum albumin, gelatin, or immunoglobulins; hydrophilic polymers such aspolyvinylpyrrolidone; amino acids such as glycine, glutamine,asparagine, histidine, arginine, or lysine; monosaccharides,disaccharides, and other carbohydrates including glucose, mannose, ordextrins; chelating agents such as EDTA; sugars such as sucrose,mannitol, trehalose or sorbitol; salt-forming counter-ions such assodium; metal complexes (e.g. Zn-protein complexes); and/or non-ionicsurfactants such as polyethylene glycol (PEG). Aqueous injectionsuspensions may contain compounds which increase the viscosity of thesuspension, such as sodium carboxymethyl cellulose, sorbitol, dextran,or the like. Optionally, the suspension may also contain suitablestabilizers or agents which increase the solubility of the compounds toallow for the preparation of highly concentrated solutions.Additionally, suspensions of the active compounds may be prepared asappropriate oily injection suspensions. Suitable lipophilic solvents orvehicles include fatty oils such as sesame oil, or synthetic fatty acidesters, such as ethyl cleats or triglycerides, or liposomes.

Active ingredients may be entrapped in microcapsules prepared, forexample, by coacervation techniques or by interfacial polymerization,for example, hydroxymethylcellulose or gelatin-microcapsules andpoly-(methylmethacylate) microcapsules, respectively, in colloidal drugdelivery systems (for example, liposomes, albumin microspheres,microemulsions, nano-particles and nanocapsules) or in macroemulsions.Such techniques are disclosed in Remington's Pharmaceutical Sciences(18th Ed. Mack Printing Company, 1990). Sustained-release preparationsmay be prepared. Suitable examples of sustained-release preparationsinclude semipermeable matrices of solid hydrophobic polymers containingthe polypeptide, which matrices are in the form of shaped articles, e.g.films, or microcapsules. In particular embodiments, prolonged absorptionof an injectable composition can be brought about by the use in thecompositions of agents delaying absorption, such as, for example,aluminum monostearate, gelatin or combinations thereof

In addition to the compositions described previously, the T cellactivating bispecific antigen binding molecules may also be formulatedas a depot preparation. Such long acting formulations may beadministered by implantation (for example subcutaneously orintramuscularly) or by intramuscular injection. Thus, for example, the Tcell activating bispecific antigen binding molecules may be formulatedwith suitable polymeric or hydrophobic materials (for example as anemulsion in an acceptable oil) or ion exchange resins, or as sparinglysoluble derivatives, for example, as a sparingly soluble salt.

Pharmaceutical compositions comprising the T cell activating bispecificantigen binding molecules of the invention may be manufactured by meansof conventional mixing, dissolving, emulsifying, encapsulating,entrapping or lyophilizing processes. Pharmaceutical compositions may beformulated in conventional manner using one or more physiologicallyacceptable carriers, diluents, excipients or auxiliaries whichfacilitate processing of the proteins into preparations that can be usedpharmaceutically. Proper formulation is dependent upon the route ofadministration chosen.

The T cell activating bispecific antigen binding molecules may beformulated into a composition in a free acid or base, neutral or saltform. Pharmaceutically acceptable salts are salts that substantiallyretain the biological activity of the free acid or base. These includethe acid addition salts, e.g., those formed with the free amino groupsof a proteinaceous composition, or which are formed with inorganic acidssuch as for example, hydrochloric or phosphoric acids, or such organicacids as acetic, oxalic, tartaric or mandelic acid. Salts formed withthe free carboxyl groups can also be derived from inorganic bases suchas for example, sodium, potassium, ammonium, calcium or ferrichydroxides; or such organic bases as isopropylamine, trimethylamine,histidine or procaine. Pharmaceutical salts tend to be more soluble inaqueous and other protic solvents than are the corresponding free baseforms.

Therapeutic Methods and Compositions

Any of the T cell activating bispecific antigen binding moleculesprovided herein may be used in therapeutic methods. T cell activatingbispecific antigen binding molecules of the invention can be used asimmunotherapeutic agents, for example in the treatment of cancers.

For use in therapeutic methods, T cell activating bispecific antigenbinding molecules of the invention would be formulated, dosed, andadministered in a fashion consistent with good medical practice. Factorsfor consideration in this context include the particular disorder beingtreated, the particular mammal being treated, the clinical condition ofthe individual patient, the cause of the disorder, the site of deliveryof the agent, the method of administration, the scheduling ofadministration, and other factors known to medical practitioners.

In one aspect, T cell activating bispecific antigen binding molecules ofthe invention for use as a medicament are provided. In further aspects,T cell activating bispecific antigen binding molecules of the inventionfor use in treating a disease are provided. In certain embodiments, Tcell activating bispecific antigen binding molecules of the inventionfor use in a method of treatment are provided. In one embodiment, theinvention provides a T cell activating bispecific antigen bindingmolecule as described herein for use in the treatment of a disease in anindividual in need thereof. In certain embodiments, the inventionprovides a T cell activating bispecific antigen binding molecule for usein a method of treating an individual having a disease comprisingadministering to the individual a therapeutically effective amount ofthe T cell activating bispecific antigen binding molecule. In certainembodiments the disease to be treated is a proliferative disorder. In aparticular embodiment the disease is cancer. In certain embodiments themethod further comprises administering to the individual atherapeutically effective amount of at least one additional therapeuticagent, e.g., an anti-cancer agent if the disease to be treated iscancer. In further embodiments, the invention provides a T cellactivating bispecific antigen binding molecule as described herein foruse in inducing lysis of a target cell, particularly a tumor cell. Incertain embodiments, the invention provides a T cell activatingbispecific antigen binding molecule for use in a method of inducinglysis of a target cell, particularly a tumor cell, in an individualcomprising administering to the individual an effective amount of the Tcell activating bispecific antigen binding molecule to induce lysis of atarget cell. An “individual” according to any of the above embodimentsis a mammal, preferably a human.

In a further aspect, the invention provides for the use of a T cellactivating bispecific antigen binding molecule of the invention in themanufacture or preparation of a medicament. In one embodiment themedicament is for the treatment of a disease in an individual in needthereof. In a further embodiment, the medicament is for use in a methodof treating a disease comprising administering to an individual havingthe disease a therapeutically effective amount of the medicament. Incertain embodiments the disease to be treated is a proliferativedisorder. In a particular embodiment the disease is cancer. In oneembodiment, the method further comprises administering to the individuala therapeutically effective amount of at least one additionaltherapeutic agent, e.g., an anti-cancer agent if the disease to betreated is cancer. In a further embodiment, the medicament is forinducing lysis of a target cell, particularly a tumor cell. In still afurther embodiment, the medicament is for use in a method of inducinglysis of a target cell, particularly a tumor cell, in an individualcomprising administering to the individual an effective amount of themedicament to induce lysis of a target cell. An “individual” accordingto any of the above embodiments may be a mammal, preferably a human.

In a further aspect, the invention provides a method for treating adisease. In one embodiment, the method comprises administering to anindividual having such disease a therapeutically effective amount of a Tcell activating bispecific antigen binding molecule of the invention. Inone embodiment a composition is administered to said individual,comprising the T cell activating bispecific antigen binding molecule ofthe invention in a pharmaceutically acceptable form. In certainembodiments the disease to be treated is a proliferative disorder. In aparticular embodiment the disease is cancer. In certain embodiments themethod further comprises administering to the individual atherapeutically effective amount of at least one additional therapeuticagent, e.g., an anti-cancer agent if the disease to be treated iscancer. An “individual” according to any of the above embodiments may bea mammal, preferably a human.

In a further aspect, the invention provides a method for inducing lysisof a target cell, particularly a tumor cell. In one embodiment themethod comprises contacting a target cell with a T cell activatingbispecific antigen binding molecule of the invention in the presence ofa T cell, particularly a cytotoxic T cell. In a further aspect, a methodfor inducing lysis of a target cell, particularly a tumor cell, in anindividual is provided. In one such embodiment, the method comprisesadministering to the individual an effective amount of a T cellactivating bispecific antigen binding molecule to induce lysis of atarget cell. In one embodiment, an “individual” is a human.

In certain embodiments the disease to be treated is a proliferativedisorder, particularly cancer. Non-limiting examples of cancers includebladder cancer, brain cancer, head and neck cancer, pancreatic cancer,lung cancer, breast cancer, ovarian cancer, uterine cancer, cervicalcancer, endometrial cancer, esophageal cancer, colon cancer, colorectalcancer, rectal cancer, gastric cancer, prostate cancer, blood cancer,skin cancer, squamous cell carcinoma, bone cancer, and kidney cancer.Other cell proliferation disorders that can be treated using a T cellactivating bispecific antigen binding molecule of the present inventioninclude, but are not limited to neoplasms located in the: abdomen, bone,breast, digestive system, liver, pancreas, peritoneum, endocrine glands(adrenal, parathyroid, pituitary, testicles, ovary, thymus, thyroid),eye, head and neck, nervous system (central and peripheral), lymphaticsystem, pelvic, skin, soft tissue, spleen, thoracic region, andurogenital system. Also included are pre-cancerous conditions or lesionsand cancer metastases. In certain embodiments the cancer is chosen fromthe group consisting of renal cell cancer, skin cancer, lung cancer,colorectal cancer, breast cancer, brain cancer, head and neck cancer. Askilled artisan readily recognizes that in many cases the T cellactivating bispecific antigen binding molecule may not provide a curebut may only provide partial benefit. In some embodiments, aphysiological change having some benefit is also consideredtherapeutically beneficial. Thus, in some embodiments, an amount of Tcell activating bispecific antigen binding molecule that provides aphysiological change is considered an “effective amount” or a“therapeutically effective amount”. The subject, patient, or individualin need of treatment is typically a mammal, more specifically a human.

In some embodiments, an effective amount of a T cell activatingbispecific antigen binding molecule of the invention is administered toa cell. In other embodiments, a therapeutically effective amount of a Tcell activating bispecific antigen binding molecule of the invention isadministered to an individual for the treatment of disease.

For the prevention or treatment of disease, the appropriate dosage of aT cell activating bispecific antigen binding molecule of the invention(when used alone or in combination with one or more other additionaltherapeutic agents) will depend on the type of disease to be treated,the route of administration, the body weight of the patient, the type ofT cell activating bispecific antigen binding molecule, the severity andcourse of the disease, whether the T cell activating bispecific antigenbinding molecule is administered for preventive or therapeutic purposes,previous or concurrent therapeutic interventions, the patient's clinicalhistory and response to the T cell activating bispecific antigen bindingmolecule, and the discretion of the attending physician. Thepractitioner responsible for administration will, in any event,determine the concentration of active ingredient(s) in a composition andappropriate dose(s) for the individual subject. Various dosing schedulesincluding but not limited to single or multiple administrations overvarious time-points, bolus administration, and pulse infusion arecontemplated herein.

The T cell activating bispecific antigen binding molecule is suitablyadministered to the patient at one time or over a series of treatments.Depending on the type and severity of the disease, about 1 μg/kg to 15mg/kg (e.g. 0.1 mg/kg-10 mg/kg) of T cell activating bispecific antigenbinding molecule can be an initial candidate dosage for administrationto the patient, whether, for example, by one or more separateadministrations, or by continuous infusion. One typical daily dosagemight range from about 1 μg/kg to 100 mg/kg or more, depending on thefactors mentioned above. For repeated administrations over several daysor longer, depending on the condition, the treatment would generally besustained until a desired suppression of disease symptoms occurs. Oneexemplary dosage of the T cell activating bispecific antigen bindingmolecule would be in the range from about 0.005 mg/kg to about 10 mg/kg.In other non-limiting examples, a dose may also comprise from about 1microgram/kg body weight, about 5 microgram/kg body weight, about 10microgram/kg body weight, about 50 microgram/kg body weight, about 100microgram/kg body weight, about 200 microgram/kg body weight, about 350microgram/kg body weight, about 500 microgram/kg body weight, about 1milligram/kg body weight, about 5 milligram/kg body weight, about 10milligram/kg body weight, about 50 milligram/kg body weight, about 100milligram/kg body weight, about 200 milligram/kg body weight, about 350milligram/kg body weight, about 500 milligram/kg body weight, to about1000 mg/kg body weight or more per administration, and any rangederivable therein. In non-limiting examples of a derivable range fromthe numbers listed herein, a range of about 5 mg/kg body weight to about100 mg/kg body weight, about 5 microgram/kg body weight to about 500milligram/kg body weight, etc., can be administered, based on thenumbers described above. Thus, one or more doses of about 0.5 mg/kg, 2.0mg/kg, 5.0 mg/kg or 10 mg/kg (or any combination thereof) may beadministered to the patient. Such doses may be administeredintermittently, e.g. every week or every three weeks (e.g. such that thepatient receives from about two to about twenty, or e.g. about six dosesof the T cell activating bispecific antigen binding molecule). Aninitial higher loading dose, followed by one or more lower doses may beadministered. However, other dosage regimens may be useful. The progressof this therapy is easily monitored by conventional techniques andassays.

The T cell activating bispecific antigen binding molecules of theinvention will generally be used in an amount effective to achieve theintended purpose. For use to treat or prevent a disease condition, the Tcell activating bispecific antigen binding molecules of the invention,or pharmaceutical compositions thereof, are administered or applied in atherapeutically effective amount. Determination of a therapeuticallyeffective amount is well within the capabilities of those skilled in theart, especially in light of the detailed disclosure provided herein.

For systemic administration, a therapeutically effective dose can beestimated initially from in vitro assays, such as cell culture assays. Adose can then be formulated in animal models to achieve a circulatingconcentration range that includes the IC₅₀ as determined in cellculture. Such information can be used to more accurately determineuseful doses in humans.

Initial dosages can also be estimated from in vivo data, e.g., animalmodels, using techniques that are well known in the art. One havingordinary skill in the art could readily optimize administration tohumans based on animal data.

Dosage amount and interval may be adjusted individually to provideplasma levels of the T cell activating bispecific antigen bindingmolecules which are sufficient to maintain therapeutic effect. Usualpatient dosages for administration by injection range from about 0.1 to50 mg/kg/day, typically from about 0.5 to 1 mg/kg/day. Therapeuticallyeffective plasma levels may be achieved by administering multiple doseseach day. Levels in plasma may be measured, for example, by HPLC.

In cases of local administration or selective uptake, the effectivelocal concentration of the T cell activating bispecific antigen bindingmolecules may not be related to plasma concentration. One having skillin the art will be able to optimize therapeutically effective localdosages without undue experimentation.

A therapeutically effective dose of the T cell activating bispecificantigen binding molecules described herein will generally providetherapeutic benefit without causing substantial toxicity. Toxicity andtherapeutic efficacy of a T cell activating bispecific antigen bindingmolecule can be determined by standard pharmaceutical procedures in cellculture or experimental animals. Cell culture assays and animal studiescan be used to determine the LD₅₀ (the dose lethal to 50% of apopulation) and the ED₅₀ (the dose therapeutically effective in 50% of apopulation). The dose ratio between toxic and therapeutic effects is thetherapeutic index, which can be expressed as the ratio LD₅₀/ED₅₀. T cellactivating bispecific antigen binding molecules that exhibit largetherapeutic indices are preferred. In one embodiment, the T cellactivating bispecific antigen binding molecule according to the presentinvention exhibits a high therapeutic index. The data obtained from cellculture assays and animal studies can be used in formulating a range ofdosages suitable for use in humans. The dosage lies preferably within arange of circulating concentrations that include the ED₅₀ with little orno toxicity. The dosage may vary within this range depending upon avariety of factors, e.g., the dosage form employed, the route ofadministration utilized, the condition of the subject, and the like. Theexact formulation, route of administration and dosage can be chosen bythe individual physician in view of the patient's condition (see, e.g.,Fingl et al., 1975, in: The Pharmacological Basis of Therapeutics, Ch.1, p. 1, incorporated herein by reference in its entirety). Theattending physician for patients treated with T cell activatingbispecific antigen binding molecules of the invention would know how andwhen to terminate, interrupt, or adjust administration due to toxicity,organ dysfunction, and the like. Conversely, the attending physicianwould also know to adjust treatment to higher levels if the clinicalresponse were not adequate (precluding toxicity). The magnitude of anadministered dose in the management of the disorder of interest willvary with the severity of the condition to be treated, with the route ofadministration, and the like. The severity of the condition may, forexample, be evaluated, in part, by standard prognostic evaluationmethods. Further, the dose and perhaps dose frequency will also varyaccording to the age, body weight, and response of the individualpatient.

Other Agents and Treatments

The T cell activating bispecific antigen binding molecules of theinvention may be administered in combination with one or more otheragents in therapy. For instance, a T cell activating bispecific antigenbinding molecule of the invention may be co-administered with at leastone additional therapeutic agent. The term “therapeutic agent”encompasses any agent administered to treat a symptom or disease in anindividual in need of such treatment. Such additional therapeutic agentmay comprise any active ingredients suitable for the particularindication being treated, preferably those with complementary activitiesthat do not adversely affect each other. In certain embodiments, anadditional therapeutic agent is an immunomodulatory agent, a cytostaticagent, an inhibitor of cell adhesion, a cytotoxic agent, an activator ofcell apoptosis, or an agent that increases the sensitivity of cells toapoptotic inducers. In a particular embodiment, the additionaltherapeutic agent is an anti-cancer agent, for example a microtubuledisruptor, an antimetabolite, a topoisomerase inhibitor, a DNAintercalator, an alkylating agent, a hormonal therapy, a kinaseinhibitor, a receptor antagonist, an activator of tumor cell apoptosis,or an antiangiogenic agent.

Such other agents are suitably present in combination in amounts thatare effective for the purpose intended. The effective amount of suchother agents depends on the amount of T cell activating bispecificantigen binding molecule used, the type of disorder or treatment, andother factors discussed above. The T cell activating bispecific antigenbinding molecules are generally used in the same dosages and withadministration routes as described herein, or about from 1 to 99% of thedosages described herein, or in any dosage and by any route that isempirically/clinically determined to be appropriate.

Such combination therapies noted above encompass combined administration(where two or more therapeutic agents are included in the same orseparate compositions), and separate administration, in which case,administration of the T cell activating bispecific antigen bindingmolecule of the invention can occur prior to, simultaneously, and/orfollowing, administration of the additional therapeutic agent and/oradjuvant. T cell activating bispecific antigen binding molecules of theinvention can also be used in combination with radiation therapy.

In another aspect, the invention provides for a bispecific antibodycomprising a) a first antigen-binding site that comprises a variableheavy chain domain (VH) of SEQ ID NO: 274 and a variable light chaindomain of SEQ ID NO: 31; and b) a second antigen-binding site thatcomprises a variable heavy chain domain (VH) of SEQ ID NO: 36 and avariable light chain domain of SEQ ID NO: 31 for use in combination withan antibody to PD-L1 or FAP-4-1BBL. In one embodiment, the bispecificantibody further comprises a third antigen-binding site that comprises avariable heavy chain domain (VH) of SEQ ID NO: 274 and a variable lightchain domain of SEQ ID NO: 31.

Articles of Manufacture

In another aspect of the invention, an article of manufacture containingmaterials useful for the treatment, prevention and/or diagnosis of thedisorders described above is provided. The article of manufacturecomprises a container and a label or package insert on or associatedwith the container. Suitable containers include, for example, bottles,vials, syringes, IV solution bags, etc. The containers may be formedfrom a variety of materials such as glass or plastic. The containerholds a composition which is by itself or combined with anothercomposition effective for treating, preventing and/or diagnosing thecondition and may have a sterile access port (for example the containermay be an intravenous solution bag or a vial having a stopper pierceableby a hypodermic injection needle). At least one active agent in thecomposition is a T cell activating bispecific antigen binding moleculeof the invention. The label or package insert indicates that thecomposition is used for treating the condition of choice. Moreover, thearticle of manufacture may comprise (a) a first container with acomposition contained therein, wherein the composition comprises a Tcell activating bispecific antigen binding molecule of the invention;and (b) a second container with a composition contained therein, whereinthe composition comprises a further cytotoxic or otherwise therapeuticagent. The article of manufacture in this embodiment of the inventionmay further comprise a package insert indicating that the compositionscan be used to treat a particular condition. Alternatively, oradditionally, the article of manufacture may further comprise a second(or third) container comprising a pharmaceutically-acceptable buffer,such as bacteriostatic water for injection (BWFI), phosphate-bufferedsaline, Ringer's solution and dextrose solution. It may further includeother materials desirable from a commercial and user standpoint,including other buffers, diluents, filters, needles, and syringes.

EXAMPLES

The following are examples of methods and compositions of the invention.It is understood that various other embodiments may be practiced, giventhe general description provided above.

General Methods

Recombinant DNA Techniques

Standard methods were used to manipulate DNA as described in Sambrook etal., Molecular cloning: A laboratory manual; Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y., 1989. The molecularbiological reagents were used according to the manufacturers'instructions. General information regarding the nucleotide sequences ofhuman immunoglobulins light and heavy chains is given in: Kabat, E. A.et al., (1991) Sequences of Proteins of Immunological Interest, 5^(th)ed., NIH Publication No. 91-3242.

DNA Sequencing

DNA Sequences were Determined by Standard Double Strand Sequencing atSynergene (Schlieren).

Gene Synthesis

Desired gene segments where required were either generated by PCR usingappropriate templates or were synthesized by Geneart AG (Regensburg,Germany) from synthetic oligonucleotides and PCR products by automatedgene synthesis. In cases where no exact gene sequence was available,oligonucleotide primers were designed based on sequences from closesthomologues and the genes were isolated by RT-PCR from RNA originatingfrom the appropriate tissue. The gene segments flanked by singularrestriction endonuclease cleavage sites were cloned into standardcloning/sequencing vectors. The plasmid DNA was purified fromtransformed bacteria and concentration determined by UV spectroscopy.The DNA sequence of the subcloned gene fragments was confirmed by DNAsequencing. Gene segments were designed with suitable restriction sitesto allow sub-cloning into the respective expression vectors. Allconstructs were designed with a 5′-end DNA sequence coding for a leaderpeptide which targets proteins for secretion in eukaryotic cells.

Isolation of Primary Human Pan T Cells from PBMCs

Peripheral blood mononuclear cells (PBMCs) were prepared by Histopaquedensity centrifugation from enriched lymphocyte preparations (buffycoats) obtained from local blood banks or from fresh blood from healthyhuman donors. Briefly, blood was diluted with sterile PBS and carefullylayered over a Histopaque gradient (Sigma, H8889). After centrifugationfor 30 minutes at 450×g at room temperature (brake switched off), partof the plasma above the PBMC containing interphase was discarded. ThePBMCs were transferred into new 50 ml Falcon tubes and tubes were filledup with PBS to a total volume of 50 ml. The mixture was centrifuged atroom temperature for 10 minutes at 400×g (brake switched on). Thesupernatant was discarded and the PBMC pellet washed twice with sterilePBS (centrifugation steps at 4° C. for 10 minutes at 350×g). Theresulting PBMC population was counted automatically (ViCell) and storedin RPMI1640 medium, containing 10% FCS and 1% L-alanyl-L-glutamine(Biochrom, K0302) at 37° C., 5% CO₂ in the incubator until assay start.

T cell enrichment from PBMCs was performed using the Pan T CellIsolation Kit II (Miltenyi Biotec #130-091-156), according to themanufacturer's instructions. Briefly, the cell pellets were diluted in40 μl cold buffer per 10 million cells (PBS with 0.5% BSA, 2 mM EDTA,sterile filtered) and incubated with 10 μl Biotin-Antibody Cocktail per10 million cells for 10 min at 4° C. 30 μl cold buffer and 20 μlAnti-Biotin magnetic beads per 10 million cells were added, and themixture incubated for another 15 min at 4° C. Cells were washed byadding 10-20× the current volume and a subsequent centrifugation step at300×g for 10 min. Up to 100 million cells were resuspended in 500 μlbuffer. Magnetic separation of unlabeled human pan T cells was performedusing LS columns (Miltenyi Biotec #130-042-401) according to themanufacturer's instructions. The resulting T cell population was countedautomatically (ViCell) and stored in AIM-V medium at 37° C., 5% CO₂ inthe incubator until assay start (not longer than 24 h).

Isolation of Primary Human Naive T Cells from PBMCs

Peripheral blood mononuclar cells (PBMCs) were prepared by Histopaquedensity centrifugation from enriched lymphocyte preparations (buffycoats) obtained from local blood banks or from fresh blood from healthyhuman donors. T-cell enrichment from PBMCs was performed using the NaiveCD8⁺ T cell isolation Kit from Miltenyi Biotec (#130-093-244), accordingto the manufacturer's instructions, but skipping the last isolation stepof CD8⁺ T cells (also see description for the isolation of primary humanpan T cells).

Isolation of Murine Pan T Cells from Splenocytes

Spleens were isolated from C57BL/6 mice, transferred into a GentleMACSC-tube (Miltenyi Biotech #130-093-237) containing MACS buffer (PBS+0.5%BSA+2 mM EDTA) and dissociated with the GentleMACS Dissociator to obtainsingle-cell suspensions according to the manufacturer's instructions.The cell suspension was passed through a pre-separation filter to removeremaining undissociated tissue particles. After centrifugation at 400×gfor 4 min at 4° C., ACK Lysis Buffer was added to lyse red blood cells(incubation for 5 min at room temperature). The remaining cells werewashed with MACS buffer twice, counted and used for the isolation ofmurine pan T cells. The negative (magnetic) selection was performedusing the Pan T Cell Isolation Kit from Miltenyi Biotec (#130-090-861),following the manufacturer's instructions. The resulting T cellpopulation was automatically counted (ViCell) and immediately used forfurther assays.

Isolation of Primary Cynomolgus PBMCs from Heparinized Blood

Peripheral blood mononuclar cells (PBMCs) were prepared by densitycentrifugation from fresh blood from healthy cynomolgus donors, asfollows: Heparinized blood was diluted 1:3 with sterile PBS, andLymphoprep medium (Axon Lab #1114545) was diluted to 90% with sterilePBS. Two volumes of the diluted blood were layered over one volume ofthe diluted density gradient and the PBMC fraction was separated bycentrifugation for 30 min at 520×g, without brake, at room temperature.The PBMC band was transferred into a fresh 50 ml Falcon tube and washedwith sterile PBS by centrifugation for 10 min at 400×g at 4° C. Onelow-speed centrifugation was performed to remove the platelets (15 minat 150×g, 4° C.), and the resulting PBMC population was automaticallycounted (ViCell) and immediately used for further assays.

Example 1 Purification of Biotinylated Folate Receptor-Fc Fusions

To generate new antibodies against human FolR1 the following antigensand screening tools were generated as monovalent Fc fusion proteins (theextracellular domain of the antigen linked to the hinge region ofFc-knob which is co-expressed with an Fc-hole molecule). The antigengenes were synthesized (Geneart, Regensburg, Germany) based on sequencesobtained from GenBank or SwissProt and inserted into expression vectorsto generate fusion proteins with Fc-knob with a C-terminal Avi-tag forin vivo or in vitro biotinylation. In vivo biotinylation was achieved byco-expression of the bacterial birA gene encoding a bacterial biotinligase during production. Expression of all genes was under control of achimeric MPSV promoter on a plasmid containing an oriP element forstable maintenance of the plasmids in EBNA containing cell lines.

For preparation of the biotinylated monomeric antigen/Fc fusionmolecules, exponentially growing suspension HEK293 EBNA cells wereco-transfected with three vectors encoding the two components of fusionprotein (knob and hole chains) as well as BirA, an enzyme necessary forthe biotinylation reaction. The corresponding vectors were used at a9.5:9.5:1 ratio (“antigen ECD-Fc knob-avi tag”: “Fc hole”: “BirA”).

For protein production in 500 ml shake flasks, 400 million HEK293 EBNAcells were seeded 24 hours before transfection. For transfection cellswere centrifuged for 5 minutes at 210 g, and supernatant was replaced bypre-warmed CD CHO medium. Expression vectors were resuspended in 20 mLof CD CHO medium containing 200 μg of vector DNA. After addition of 540μL of polyethylenimine (PEI), the solution was mixed for 15 seconds andincubated for 10 minutes at room temperature. Afterwards, cells weremixed with the DNA/PEI solution, transferred to a 500 mL shake flask andincubated for 3 hours at 37° C. in an incubator with a 5% CO₂atmosphere. After the incubation, 160 mL of F17 medium was added andcells were cultured for 24 hours. One day after transfection, 1 mMvalproic acid and 7% Feed 1 (Lonza) were added to the culture. Theproduction medium was also supplemented with 100 μM biotin. After 7 daysof culturing, the cell supernatant was collected by spinning down cellsfor 15 min at 210 g. The solution was sterile filtered (0.22 μm filter),supplemented with sodium azide to a final concentration of 0.01% (w/v),and kept at 4° C.

Secreted proteins were purified from cell culture supernatants byaffinity chromatography using Protein A, followed by size exclusionchromatography. For affinity chromatography, the supernatant was loadedon a HiTrap ProteinA HP column (CV=5 mL, GE Healthcare) equilibratedwith 40 mL 20 mM sodium phosphate, 20 mM sodium citrate pH 7.5. Unboundprotein was removed by washing with at least 10 column volumes of 20 mMsodium phosphate, 20 mM sodium citrate pH 7.5. The bound protein waseluted using a linear pH-gradient created over 20 column volumes of 20mM sodium citrate, 100 mM sodium chloride, 100 mM glycine, pH 3.0. Thecolumn was then washed with 10 column volumes of 20 mM sodium citrate,100 mM sodium chloride, 100 mM glycine, pH 3.0.

pH of collected fractions was adjusted by adding 1/10 (v/v) of 0.5 Msodium phosphate, pH 8.0. The protein was concentrated and filteredprior to loading on a HiLoad Superdex 200 column (GE Healthcare)equilibrated with 20 mM histidine, 140 mM sodium chloride, pH 6.0.

The protein concentration was determined by measuring the opticaldensity (OD) at 280 nm, using the molar extinction coefficientcalculated on the basis of the amino acid sequence. Purity and molecularweight of the FolR1-Fc-fusion was analyzed by SDS capillaryelectrophoresis in the presence and absence of a reducing agentfollowing the manufacturer instructions (instrument Caliper LabChipGX,Perkin Elmer). The aggregate content of samples was analyzed using aTSKgel G3000 SW XL analytical size-exclusion column (Tosoh) equilibratedin 25 mM K2HPO4, 125 mM NaCl, 200 mM L-arginine monohydrochloride, 0.02%(w/v) NaN3, pH 6.7 running buffer at 25° C. Purified antigen-Fc-fusionproteins were analyzed by surface plasmon resonance assays usingcommercially available antibodies to confirm correct and naturalconformation of the antigens (data not shown).

TABLE 1Antigens produced for isolation, selection and counter selection ofhuman FolR1 antibodies ECD Accession Seq ID Antigen (aa) number SequenceNo human 25-234 P15328 RIAWARTELLNVCMNAKHHKEKPGPEDKLHEQCRPWR 227 FolR1KNACCSTNTSQEAHKDVSYLYRFNWNHCGEMAPACKRHFIQDTCLYECSPNLGPWIQQVDQSWRKERVLNVPLCKEDCEQWWEDCRTSYTCKSNWHKGWNWTSGFNKCAVGAACQPFHFYFPTPTVLCNEIWTHSYKVSNYSRGSGRC IQMWFDPAQGNPNEEVARFYAAAM human17-230 P14207 TMCSAQDRTDLLNVCMDAKHHKTKPGPEDKLHDQCSP 228 FolR2WKKNACCTASTSQELHKDTSRLYNFNWDHCGKMEPACKRHFIQDTCLYECSPNLGPWIQQVNQSWRKERFLDVPLCKEDCQRWWEDCHTSHTCKSNWHRGWDWTSGVNKCPAGALCRTFESYFPTPAALCEGLWSHSYKVSNYSRGSG RCIQMWFDSAQGNPNEEVARFYAAAMHVNhuman 24-243 P41439 SARARTDLLNVCMNAKHHKTQPSPEDELYGQCSPWKK 229 FolR3NACCTASTSQELHKDTSRLYNFNWDHCGKMEPTCKRHFIQDSCLYECSPNLGPWIRQVNQSWRKERILNVPLCKEDCERWWEDCRTSYTCKSNWHKGWNWTSGINECPAGALCSTFESYFPTPAALCEGLWSHSFKVSNYSRGSGRCIQMWFDSAQGNPNEEVAKFYAAAMNAGAPSRGIIDS murine 25-232 P35846TRARTELLNVCMDAKHHKEKPGPEDNLHDQCSPWKTN 230 FolR1SCCSTNTSQEAHKDISYLYRENWNHCGTMTSECKRHEIQDTCLYECSPNLGPWIQQVDQSWRKERILDVPLCKEDCQQWWEDCQSSFTCKSNWHKGWNWSSGHNECPVGASCHPFTFYFPTSAALCEEIWSHSYKLSNYSRGSGRCIQ MWFDPAQGNPNEEVARFYAEAMS cynomolgus25-234 G7PR14 EAQTRTARARTELLNVCMNAKHHKEKPGPEDKLHEQC 231 FolR1RPWKKNACCSTNTSQEAHKDVSYLYRFNWNHCGEMAPACKRHFIQDTCLYECSPNLGPWIQQVDQSWRKERVLNVPLCKEDCERWWEDCRTSYCKSNWHKGWNWTSGFNKCPVGAACQPFHFYFPTPTVLCNEIWTYSYKVSNYSRGS GRCIQMWFDPAQGNPNEEVARFYAAAMS

TABLE 2 Summary of the yield and final monomer content of theFolR-Fc-fusions. Monomer [%] Antigen (SEC) Yield huFolR1 100 30 mg/LcyFolR1 100 32 mg/L muFolR1 100 31 mg/L huFolR2 100 16 mg/L huFolR3 9538 mg/L

Example 2 Generation of Common Light Chain with CD3ε Specificity

The T cell activating bispecific molecules described herein comprise atleast one CD3 binding moiety. This moiety can be generated by immunizinglaboratory animals, screening phage library or using known anti-CD3antibodies. The common light chain with CD3ε specificity was generatedby humanizing the light chain of a murine parental anti-CD3ε antibody(CH2527). For humanization of an antibody of non-human origin, the CDRresidues from the non-human antibody (donor) have to be transplantedonto the framework of a human (acceptor) antibody. Generally, acceptorframework sequences are selected by aligning the sequence of the donorto a collection of potential acceptor sequences and choosing one thathas either reasonable homology to the donor, or shows similar aminoacids at some positions critical for structure and activity. In thepresent case, the search for the antibody acceptor framework wasperformed by aligning the mouse VL-domain sequence of the parentalantibody to a collection of human germline sequences and choosing thehuman sequence that showed high sequence identity. Surprisingly, a goodmatch in terms of framework sequence homology was found in a ratherinfrequent human light chain belonging to the V-domain family 7 of thelambda type, more precisely, hVL_7_46 (IMGT nomenclature, GenBank AccNo. Z73674). This infrequent human light chain was subsequently chosenas acceptor framework for humanization of the light chain of CH2527. Thethree complementarity determining regions (CDRs) of the mouse lightchain variable domain were grafted onto this acceptor framework. Sincethe framework 4 region is not part of the variable region of thegermline V-gene, the alignment for this region (J-element) was doneindividually. Hence the IGLJ3-02 sequence was chosen for humanization ofthis light chain.

Thirteen humanized variants were generated (CH2527-VL7_46-1 toVL7_46-10, VL7_46-12 to VL7_46-14). These differ in framework residues(and combinations thereof) that were back-mutated to the murine V-domainsequence or in CDR-residues (Kabat definition) that could be keptidentical to the human germline sequence. The following frameworkresidues outside the CDRs were back-mutated to the murine residues inthe final humanized VL-domain variant VL7_46-13 (murine residueslisted): V36, E38, F44, G46, G49, and G57, respectively. The humanJ-element IGLJ3-02 was 100% identical to the J-element of the murineparental antibody.

Example 3 SPR Assessment of Humanized Variants with CD3ε Specificity

Humanized VL variants were assessed as chimera in a 2+1 classical format(FIG. 1D), i.e. humanized light chain V-domains were paired with murineheavy chain V-domains. SPR assessment was carried out on a ProteOn XPR36instrument (Bio-Rad). More precisely, the variants were captureddirectly from the culture supernatant on an anti-Fab derivatized GLMsensorchip (Goat Anti-Human IgG, F(ab′)2 Fragment Specific, JacksonImmunoResearch) in vertical orientation. The following analytes weresubsequently injected horizontally as single concentrations to assessbinding to human and cynomolgus CD3ε: 3 μM hu CD3ε(−1-26)-Fc(knob)-avi(ID807) and 2.5 μM cy CD3ε-(−1-26)-Fc(knob)-Avi-Fc(hole) (ID873),respectively. Binding responses were qualitatively compared to bindingof the murine control construct and graded+(comparable bindingobserved), +/−(reduced binding observed) and −(no binding observed). Thecapture antibody was regenerated after each cycle of ligand capture andanalyte binding and the murine construct was re-injected at the end ofthe study to confirm the activity of the capture surface. The resultsare summarized in Table 3.

TABLE 3 Qualitative binding assessment based on SPR for the humanizedlight chain variants combined with the murine heavy chain of CH2527.Only the humanized light chain variant that was finally chosen,CH2527-VL7_46-13, highlighted in bold letters, exhibited comparablebinding to human and cynomolgus CD3ε. humanized VL variant binding toCD3ε murine_CH2527-VL + CH2527-VL7_46-1 − CH2527-VL7_46-2 −CH2527-VL7_46-3 − CH2527-VL7_46-4 − CH2527-VL7_46-5 − CH2527-VL7_46-6 −CH2527-VL7_46-7 − CH2527-VL7_46-8 − CH2527-VL7_46-9 − CH2527-VL7_46-10 −CH2527-VL7_46-12 +/− CH2527-VL7_46-13 + CH2527-VL7_46-14 −

Example 4 Properties of Humanized Common Light Chain with CD3εSpecificity

The light chain V-domain variant that was chosen for the humanized leadmolecule is VL7_46-13. The degree of humanness, i.e. the sequencehomology of the humanized V-domain to the human germline V-domainsequence was determined. For VL7_46-13, the overall sequence identitywith the closest human germline homolog is 65% before humanization and80% afterwards. Omitting the CDR regions, the sequence identity is 92%to the closest human germline homolog. As can be seen from Table 3,VL7_46-13 is the only humanized VL variant out of a panel of 13 variantsthat showed comparable binding to the parental murine antibody and alsoretained its cross-reactivity to cynomolgus CD3ε. This result indicatesthat it was not trivial to humanize the murine VL-domain without losingbinding affinity to CD3ε which required several back-mutations to murineframework residues (in particular G46) while retaining G24 in CDR1. Inaddition, this result shows that the VL-domain plays a crucial role intarget recognition. Importantly, the humanized VL-domain VL7_46-13 basedon an infrequent human germline belonging to the V-domain family 7 ofthe lambda type and retaining affinity and specificity for CD3ε, is alsosuitable to be used as a common light chain in phage-displayed antibodylibraries of the Fab-format and enables successful selection for novelspecificities which greatly facilitates the generation and production ofbispecific molecules binding to CD3ε and e.g. a tumor target and sharingthe same ‘common’ light chain.

Example 5 Generation of a Phage Displayed Antibody Library Using a HumanGerm-Line Common Light Chain Derived from HVK1-39

Several approaches to generate bispecific antibodies that resemble fulllength human IgG utilize modifications in the Fc region that induceheterodimerization of two distinct heavy chains. Such examples includeknobs-into-holes (Merchant et al., Nat Biotechnol. 1998 July;16(7):677-81) SEED (Davis et al., Protein Eng Des Sel. 2010 April;23(4):195-202) and electrostatic steering technologies (Gunasekaran etal., J Biol Chem. 2010 Jun. 18; 285(25):19637-46). Although theseapproaches enable effective heterodimerization of two distinct heavychains, appropriate pairing of cognate light and heavy chains remains aproblem. Usage of a common light chain (LC) can solve this issue(Merchant, et al. Nat Biotech 16, 677-681 (1998)).

Here, we describe the generation of an antibody library for the displayon a M13 phage. Essentially, we designed a multi framework library forthe heavy chain with one constant (or “common”) light chain. Thislibrary is designed for generating multispecific antibodies without theneed to use sophisticated technologies to avoid light chain mispairing.

By using a common light chain the production of these molecules can befacilitated as no mispairing occurs any longer and the isolation of ahighly pure bispecific antibody is facilitated. As compared to otherformats the use of Fab fragments as building blocks as opposed to e.g.the use of scFv fragments results in higher thermal stability and thelack of scFv aggregation and intermolecular scFv formation.

Library Generation

In the following the generation of an antibody library for the displayon M13 phage is described. Essentially, we designed a multi frameworklibrary for the heavy chain with one constant (or “common”) light chain.

We used these heavy chains in the library (GenBank Accession Numbers inbrackets):

IGHV1-46*01 (X92343) (SEQ ID NO:104),

IGHV1-69*06 (L22583), (SEQ ID NO:105)

IGHV3-15*01 (X92216), (SEQ ID NO:106)

IGHV3-23*01 (M99660), (SEQ ID NO:107)

IGHV4-59*01 (AB019438), (SEQ ID NO:108)

IGHV5-51*01 (M99686), (SEQ ID NO:109)

All heavy chains use the IGHJ2 as J-element, except the IGHV1-69*06which uses IGHJ6 sequence.

The design of the randomization included the CDR-H1, CDR-H2, and CDR-H3.For CDR-H1 and CDR-H2 a “soft” randomization strategy was chosen, andthe randomization oligonucleotides were such that the codon for theamino acid of the germ-line sequence was present at 50%. All other aminoacids, except cysteine, were summing up for the remaining 50%. InCDR-H3, where no germ-line amino acid is present due to the presence ofthe genetic D-element, oligonucleotides were designed that allow for theusage of randomized inserts between the V-element and the J-element of 4to 9 amino acids in length. Those oligonucleotides contained in theirrandomized part e.g. The three amino acids G/Y/S are present to 15%each, those amino acids A/D/T/R/P/L/V/N/W/F/I/E are present to 4.6%each.

Exemplary methods for generation of antibody libraries are described inHoogenboom et al., Nucleic Acids Res. 1991, 19, 4133-413; Lee et., al J.Mol. Biol. (2004) 340, 1073-1093.

The light chain is derived from the human sequence hVK1-39, and is usedin an unmodified and non-randomized fashion. This will ensure that thesame light chain can be used for other projects without additionalmodifications.

Exemplary Library Selection:

Selections with all affinity maturation libraries are carried out insolution according to the following procedure using a monomeric andbiotinylated extracellular domain of a target antigen X. 1. 10̂12phagemid particles of each library are bound to 100 nM biotinylatedsoluble antigen for 0.5 h in a total volume of 1 ml. 2. Biotinylatedantigen is captured and specifically bound phage particles are isolatedby addition of ˜5×10̂7 streptavidin-coated magnetic beads for 10 min. 3.Beads are washed using 5-10×1 ml PBS/Tween20 and 5-10×1 ml PBS. 4.Elution of phage particles is done by addition of 1 ml 100 mM TEA(triethylamine) for 10 min and neutralization by addition of 500 ul 1MTris/HCl pH 7.4 and 5. Re-infection of exponentially growing E. coli TG1bacteria, infection with helper phage VCSM13 and subsequent PEG/NaClprecipitation of phagemid particles is applied in subsequent selectionrounds. Selections are carried out over 3-5 rounds using either constantor decreasing (from 10A-7M to 2×10̂−9M) antigen concentrations. In round2, capture of antigen/phage complexes is performed using neutravidinplates instead of streptavidin beads. All binding reactions aresupplemented either with 100 nM bovine serum albumin, or with non-fatmilk powder in order to compete for unwanted clones arising from meresticky binding of the antibodies to the plastic support.

Selections are being carried out over three or four rounds usingdecreasing antigen concentrations of the antigen starting from 100 nMand going down to 5 nM in the final selection round. Specific bindersare defined as signals ca. 5× higher than background and are identifiedby ELISA. Specific binders are identified by ELISA as follows: 100 μl of10 nM biotinylated antigen per well are coated on neutravidin plates.Fab-containing bacterial supernatants are added and binding Fabs aredetected via their Flag-tags by using an anti-Flag/HRP secondaryantibody. ELISA-positive clones are bacterially expressed as soluble Fabfragments in 96-well format and supernatants are subjected to a kineticscreening experiment by SPR-analysis using ProteOn XPR36 (BioRad).Clones expressing Fabs with the highest affinity constants areidentified and the corresponding phagemids are sequenced. For furthercharacterization, the Fab sequences are amplified via PCR from thephagemid and cloned via appropriate restriction sites into human IgG1expression vectors for mammalian production.

Generation of a Phage Displayed Antibody Library Using a Humanized CD3εSpecific Common Light Chain

Here, the generation of an antibody library for the display on M13 phageis described. Essentially, we designed a multi framework library for theheavy chain with one constant (or “common”) light chain. This librarywas designed for the generation of Fc-containing, but FcgR bindinginactive T cell bispecific antibodies of IgG1 P329G LALA or IgG4 SPLE PGisotype in which one or two Fab recognize a tumor surface antigenexpressed on a tumor cell whereas the remaining Fab arm of the antibodyrecognizes CD3e on a T cell.

Library Generation

In the following the generation of an antibody library for the displayon M13 phage is described. Essentially, we designed a multi frameworklibrary for the heavy chain with one constant (or “common”) light chain.This library is designed solely for the generation of Fc-containing, butFcgR binding inactive T cell bispecific antibodies of IgG1 P329G LALA orIgG4 SPLE PG isotype.

Diversity was introduced via randomization oligonucleotides only in theCDR3 of the different heavy chains. Methods for generation of antibodylibraries are well known in the art and are described in (Hoogenboom etal., Nucleic Acids Res. 1991, 19, 4133-413; or in: Lee et., al J. Mol.Biol. (2004) 340, 1073-1093).

We used these heavy chains in the library:

IGHV1-46*01 (X92343), (SEQ ID NO:104)

IGHV1-69*06 (L22583), (SEQ ID NO:105)

IGHV3-15*01 (X92216), (SEQ ID NO:106)

IGHV3-23*01 (M99660), (SEQ ID NO:107)

IGHV4-59*01 (AB019438), (SEQ ID NO:108)

IGHV5-51*01 (M99686), (SEQ ID NO:109)

We used the light chain derived from the humanized human and CynomolgusCD3ε specific antibody CH2527 in the library: (VL7_46-13; SEQ IDNO:112). This light chain was not randomized and used without anyfurther modifications in order to ensure compatibility with differentbispecific binders.

All heavy chains use the IGHJ2 as J-element, except the IGHV1-69*06which uses IGHJ6 sequence. The design of the randomization focused onthe CDR-H3 only, and PCR oligonucleotides were designed that allow forthe usage of randomized inserts between the V-element and the J-elementof 4 to 9 amino acids in length.

Example 6 Selection of Antibody Fragments from Common Light ChainLibraries (Comprising Light Chain with CD3ε Specificity) to FolR1

The antibodies 16A3, 15A1, 18D3, 19E5, 19A4, 15H7, 15B6, 16D5, 15E12,21D1, 16F12, 21A5, 21G8, 19H3, 20G6, and 20H7 comprising the commonlight chain VL7_46-13 with CD3ε specificity were obtained by phagedisplay selections against different species (human, cynomolgus andmurine) of FolR1. Clones 16A3, 15A1, 18D3, 19E5, 19A4, 15H7, 15B6, 21D1,16F12, 19H3, 20G6, and 20H7 were selected from a sub-library in whichthe common light chain was paired with a heavy chain repertoire based onthe human germline VH1_46. In this sub-library, CDR3 of VH1_46 has beenrandomized based on 6 different CDR3 lengths. Clones 16D5, 15E12, 21A5,and 21G8 were selected from a sub-library in which the common lightchain was paired with a heavy chain repertoire based on the humangermline VH3_15. In this sub-library, CDR3 of VH3_15 has been randomizedbased on 6 different CDR3 lengths. In order to obtain speciescross-reactive (or murine FolR1-reactive) antibodies, the differentspecies of FolR1 were alternated (or kept constant) in different waysover 3 rounds of biopanning: 16A3 and 15A1 (human-cynomolgus-humanFolR1); 18D3 (cynomolgus-human-murine FolR1); 19E5 and 19A4 (3 roundsagainst murine FolR1); 15H7, 15B6, 16D5, 15E12, 21D1, 16F12, 21A5, 21G8(human-cynomolgus-human FolR1); 19H3, 20G6, and 20H7 (3 rounds againstmurine FolR1).

Human, murine and cynomolgus FolR1 as antigens for the phage displayselections as well as ELISA- and SPR-based screenings were transientlyexpressed as N-terminal monomeric Fc-fusion in HEK EBNA cells and invivo site-specifically biotinylated via co-expression of BirA biotinligase at the avi-tag recognition sequence located at the C-terminus ofthe Fc portion carrying the receptor chain (Fc knob chain). In order toassess the specificity to FolR1, two related receptors, human FolR2 andFolR3 were generated in the same way.

Selection rounds (biopanning) were performed in solution according tothe following pattern:

1. Pre-clearing of −10¹² phagemid particles on maxisorp plates coatedwith 10 ug/ml of an unrelated human IgG to deplete the libraries ofantibodies recognizing the Fc-portion of the antigen.

2. Incubating the non-Fc-binding phagemid particles with 100 nMbiotinylated human, cynomolgus, or murine FolR1 for 0.5 h in thepresence of 100 nM unrelated non-biotinylated Fc knob-into-holeconstruct for further depletion of Fc-binders in a total volume of 1 ml.

3. Capturing the biotinylated FolR1 and attached specifically bindingphage by transfer to 4 wells of a neutravidin pre-coated microtiterplate for 10 min (in rounds 1 & 3).

4. Washing the respective wells using 5×PBS/Tween20 and 5×PBS.

5. Eluting the phage particles by addition of 250 ul 100 mM TEA(triethylamine) per well for 10 min and neutralization by addition of500 ul 1 M Tris/HCl pH 7.4 to the pooled eluates from 4 wells.

6. Post-clearing of neutralized eluates by incubation on neutravidinpre-coated microtiter plate with 100 nM biotin-captured FolR2 or FolR3for final removal of Fc- and unspecific binders.

7. Re-infection of log-phase E. coli TG1 cells with the supernatant ofeluted phage particles, infection with helperphage VCSM13, incubation ona shaker at 30° C. over night and subsequent PEG/NaCl precipitation ofphagemid particles to be used in the next selection round.

Selections were carried out over 3 rounds using constant antigenconcentrations of 100 nM. In round 2, in order to avoid enrichment ofbinders to neutravidin, capture of antigen:phage complexes was performedby addition of 5.4×10⁷ streptavidin-coated magnetic beads. Specificbinders were identified by ELISA as follows: 100 ul of 25 nMbiotinylated human, cynomolgus, or murine FolR1 and 10 ug/ml of humanIgG were coated on neutravidin plates and maxisorp plates, respectively.Fab-containing bacterial supernatants were added and binding Fabs weredetected via their Flag-tags using an anti-Flag/HRP secondary antibody.Clones exhibiting signals on human FolR1 and being negative on human IgGwere short-listed for further analyses and were also tested in a similarfashion against the remaining two species of FolR1. They werebacterially expressed in a 0.5 liter culture volume, affinity purifiedand further characterized by SPR-analysis using BioRad's ProteOn XPR36biosensor.

Affinities (K_(D)) of selected clones were measured by surface plasmonresonance (SPR) using a ProteOn XPR36 instrument (Biorad) at 25° C. withbiotinylated human, cynomolgus, and murine FolR1 as well as human FolR2and FolR3 (negative controls) immobilized on NLC chips by neutravidincapture. Immobilization of antigens (ligand): Recombinant antigens werediluted with PBST (10 mM phosphate, 150 mM sodium chloride pH 7.4,0.005% Tween 20) to 10 μg/ml, then injected at 30 μl/minute in verticalorientation. Injection of analytes: For ‘one-shot kinetics’measurements, injection direction was changed to horizontal orientation,two-fold dilution series of purified Fab (varying concentration ranges)were injected simultaneously along separate channels 1-5, withassociation times of 200 s, and dissociation times of 600 s. Buffer(PBST) was injected along the sixth channel to provide an “in-line”blank for referencing. Association rate constants (k_(on)) anddissociation rate constants (k_(off)) were calculated using a simpleone-to-one Langmuir binding model in ProteOn Manager v3.1 software bysimultaneously fitting the association and dissociation sensorgrams. Theequilibrium dissociation constant (K_(D)) was calculated as the ratiok_(off)/k_(on). Table 4 lists the equilibrium dissociation constants(K_(D)) of the selected clones specific for FolR1.

TABLE 4 Equilibrium dissociation constants (KD) for anti-FolR1antibodies (Fab-format) selected by phage display from common lightchain sub-libraries comprising VL7_46-13, a humanized light chainspecific for CD3ε. KD in nM. huFolR1 muFolR1 huFolR2 huFolR3 Clone [nM]cyFolR1 [nM] [nM] [nM] [nM] 16A3 21.7 18 very weak no binding no binding15A1 30.9 17.3 very weak no binding no binding 18D3 93.6 40.2 very weakno binding no binding 19E5 522 276 19.4 no binding no binding 19A4 20504250 43.1 no binding no binding 15H7 13.4 72.5 no binding no binding nobinding 15B6 19.1 13.9 no binding no binding no binding 16D5 39.5 114 nobinding no binding no binding 15E12 55.7 137 no binding no binding nobinding 21D1 62.6 32.1 no binding no binding no binding 16F12 68 90.9 nobinding no binding no binding 21A5 68.8 131 no binding no binding nobinding 21G8 130 261 no binding no binding no binding 19H3 no binding nobinding 89.7 no binding no binding 20G6 no binding no binding 78.5 nobinding no binding

Example 7 Selection of Antibody Fragments from Generic Multi-FrameworkLibraries to FolR1

The antibodies 11F8, 36F2, 9D11, 5D9, 6B6, and 14E4 were obtained byphage display selections based on generic multi-framework sub-librariesagainst different species (human, cynomolgus and murine) of FolR1. Inthese multi-framework sub-libraries, different VL-domains withrandomized CDR3 (3 different lengths) are paired with differentVH-domains with randomized CDR3 (6 different lengths). The selectedclones are of the following VL/VH pairings: 11F8 (Vk_1_5/VH_1_69), 36F2(Vk_3_20/VH_1_46), 9D11 (Vk2D_28/VH1_46), 5D9 (Vk3_20/VH1_46), 6B6(Vk3_20/VH1_46), and 14E4 (Vk3_20/VH3_23). In order to obtain speciescross-reactive (or murine FolR1-reactive) antibodies, the differentspecies of FolR1 were alternated (or kept constant) in different waysover 3 or 4 rounds of biopanning: 11F8 (cynomolgus-murine-human FolR1);36F2 (human-murine-cynomolgus-murine FolR1); 9D11(cynomolgus-human-cynomolgus FolR1); 5D9 (human-cynomolgus-human FolR1);6B6 (human-cynomolgus-human FolR1) and 14E4 (3 rounds against murineFolR1).

Human, murine and cynomolgus FolR1 as antigens for the phage displayselections as well as ELISA- and SPR-based screenings were transientlyexpressed as N-terminal monomeric Fc-fusion in HEK EBNA cells and invivo site-specifically biotinylated via co-expression of BirA biotinligase at the avi-tag recognition sequence located at the C-terminus ofthe Fc portion carrying the receptor chain (Fc knob chain). In order toassess the specificity to FolR1, two related receptors, human FolR2 andFolR3 were generated in the same way.

Selection rounds (biopanning) were performed in solution according tothe following pattern:

1. Pre-clearing of ˜10¹² phagemid particles on maxisorp plates coatedwith 10 ug/ml of an unrelated human IgG to deplete the libraries ofantibodies recognizing the Fc-portion of the antigen.

2. Incubating the non-Fc-binding phagemid particles with 100 nMbiotinylated human, cynomolgus, or murine FolR1 for 0.5 h in thepresence of 100 nM unrelated non-biotinylated Fc knob-into-holeconstruct for further depletion of Fc-binders in a total volume of 1 ml.

3. Capturing the biotinylated FolR1 and attached specifically bindingphage by transfer to 4 wells of a neutravidin pre-coated microtiterplate for 10 min (in rounds 1 & 3).

4. Washing the respective wells using 5×PBS/Tween20 and 5×PBS.

5. Eluting the phage particles by addition of 250 ul 100 mM TEA(triethylamine) per well for 10 min and neutralization by addition of500 ul 1 M Tris/HCl pH 7.4 to the pooled eluates from 4 wells.

6. Post-clearing of neutralized eluates by incubation on neutravidinpre-coated microtiter plate with 100 nM biotin-captured FolR2 or FolR3for final removal of Fc- and unspecific binders.

7. Re-infection of log-phase E. coli TG1 cells with the supernatant ofeluted phage particles, infection with helperphage VCSM13, incubation ona shaker at 30° C. over night and subsequent PEG/NaCl precipitation ofphagemid particles to be used in the next selection round.

Selections were carried out over 3 rounds using constant antigenconcentrations of 100 nM. In round 2 and 4, in order to avoid enrichmentof binders to neutravidin, capture of antigen:phage complexes wasperformed by addition of 5.4×10⁷ streptavidin-coated magnetic beads.Specific binders were identified by ELISA as follows: 100 ul of 25 nMbiotinylated human, cynomolgus, or murine FolR1 and 10 ug/ml of humanIgG were coated on neutravidin plates and maxisorp plates, respectively.

Fab-containing bacterial supernatants were added and binding Fabs weredetected via their Flag-tags using an anti-Flag/HRP secondary antibody.Clones exhibiting signals on human FolR1 and being negative on human IgGwere short-listed for further analyses and were also tested in a similarfashion against the remaining two species of FolR1. They werebacterially expressed in a 0.5 liter culture volume, affinity purifiedand further characterized by SPR-analysis using BioRad's ProteOn XPR36biosensor.

Affinities (K_(D)) of selected clones were measured by surface plasmonresonance (SPR) using a ProteOn XPR36 instrument (Biorad) at 25° C. withbiotinylated human, cynomolgus, and murine FolR1 as well as human FolR2and FolR3 (negative controls) immobilized on NLC chips by neutravidincapture. Immobilization of antigens (ligand): Recombinant antigens werediluted with PBST (10 mM phosphate, 150 mM sodium chloride pH 7.4,0.005% Tween 20) to 10 μg/ml, then injected at 30 μl/minute in verticalorientation. Injection of analytes: For ‘one-shot kinetics’measurements, injection direction was changed to horizontal orientation,two-fold dilution series of purified Fab (varying concentration ranges)were injected simultaneously along separate channels 1-5, withassociation times of 150 or 200 s, and dissociation times of 200 or 600s, respectively. Buffer (PBST) was injected along the sixth channel toprovide an “in-line” blank for referencing. Association rate constants(k_(on)) and dissociation rate constants (k_(off)) were calculated usinga simple one-to-one Langmuir binding model in ProteOn Manager v3.1software by simultaneously fitting the association and dissociationsensorgrams. The equilibrium dissociation constant (K_(D)) wascalculated as the ratio k_(off)/k_(on). Table 5 lists the equilibriumdissociation constants (K_(D)) of the selected clones specific forFolR1.

TABLE 5 Equilibrium dissociation constants (K_(D)) for anti-FolR1antibodies (Fab-format) selected by phage display from genericmulti-framework sub-libraries. K_(D) in nM. K_(D) (nM) Clone huFolR1cyFolR1 muFolR1 huFolR2 huFolR3 11F8 632 794 1200 no binding no binding36F2 1810 1640 737 no binding no binding 9D11 8.64 5.29 no binding nobinding no binding 5D9 8.6 5.9 no binding no binding no binding 6B6 14.59.4 no binding no binding no binding 14E4 no binding no binding 6.09 nobinding no binding

Example 8 Production and Purification of Novel FolR1 Binders in IgG andT-Cell Bispecific Formats

To identify FolR1 binders which are able to induce T-cell dependentkilling of selected target cells the antibodies isolated from a commonlight chain- or Fab-library were converted into the corresponding humanIgG1 format. In brief, the variable heavy and variable light chains ofunique FolR1 binders from phage display were amplified by standard PCRreactions using the Fab clones as the template. The PCR products werepurified and inserted (either by restriction endonuclease and ligasebased cloning, or by ‘recombineering’ using the InFusion kit fromInvitrogen) into suitable expression vectors in which they are fused tothe appropriate human constant heavy or human constant light chain. Theexpression cassettes in these vectors consist of a chimeric MPSVpromoter and a synthetic polyadenylation site. In addition, the plasmidscontain the oriP region from the Epstein Barr virus for the stablemaintenance of the plasmids in HEK293 cells harboring the EBV nuclearantigen (EBNA). After PEI mediated transfection the antibodies weretransiently produced in HEK293 EBNA cells and purified by standardProteinA affinity chromatography followed by size exclusionchromatography as described:

Transient Transfection and Production

All (bispecific) antibodies (if not obtained from a commercial source)used herein were transiently produced in HEK293 EBNA cells using a PEImediated transfection procedure for the required vectors as describedbelow. HEK293 EBNA cells are cultivated in suspension serum free in CDCHO culture medium. For the production in 500 ml shake flask 400 millionHEK293 EBNA cells are seeded 24 hours before transfection (foralternative scales all amounts were adjusted accordingly). Fortransfection cells are centrifuged for 5 min by 210×g, supernatant isreplaced by pre-warmed 20 ml CD CHO medium. Expression vectors are mixedin 20 ml CD CHO medium to a final amount of 200 μg DNA. After additionof 540 μl PEI solution is vortexed for 15 s and subsequently incubatedfor 10 min at room temperature. Afterwards cells are mixed with theDNA/PEI solution, transferred to a 500 ml shake flask and incubated for3 hours by 37° C. in an incubator with a 5% CO2 atmosphere. Afterincubation time 160 ml F17 medium is added and cell are cultivated for24 hours. One day after transfection 1 mM valporic acid and 7% Feed 1 isadded. After 7 days cultivation supernatant is collected forpurification by centrifugation for 15 min at 210×g, the solution issterile filtered (0.22 μm filter) and sodium azide in a finalconcentration of 0.01% w/v is added, and kept at 4° C. After productionthe supernatants were harvested and the antibody containing supernatantswere filtered through 0.22 μm sterile filters and stored at 4° C. untilpurification.

Antibody Purification

All molecules were purified in two steps using standard procedures, suchas protein A affinity purification (Äkta Explorer) and size exclusionchromatography. The supernatant obtained from transient production wasadjusted to pH 8.0 (using 2 M TRIS pH 8.0) and applied to HiTrap PA FF(GE Healthcare, column volume (cv)=5 ml) equilibrated with 8 columnvolumes (cv) buffer A (20 mM sodium phosphate, 20 mM sodium citrate, pH7.5). After washing with 10 cv of buffer A, the protein was eluted usinga pH gradient to buffer B (20 mM sodium citrate pH 3, 100 mM NaCl, 100mM glycine) over 12 cv. Fractions containing the protein of interestwere pooled and the pH of the solution was gently adjusted to pH 6.0(using 0.5 M Na₂HPO₄ pH 8.0). Samples were concentrated to 2 ml usingultra-concentrators (Vivaspin 15R 30.000 MWCO HY, Sartorius) andsubsequently applied to a HiLoad™ 16/60 Superdex™ 200 preparative grade(GE Healthcare) equilibrated with 20 mM Histidine, pH 6.0, 140 mM NaCl,0.01% Tween-20. The aggregate content of eluted fractions was analyzedby analytical size exclusion chromatography. Therefore, 30 μl of eachfraction was applied to a TSKgel G3000 SW XL analytical size-exclusioncolumn (Tosoh) equilibrated in 25 mM K₂HPO₄, 125 mM NaCl, 200 mML-arginine monohydrochloride, 0.02% (w/v) NaN₃, pH 6.7 running buffer at25° C. Fractions containing less than 2% oligomers were pooled andconcentrated to final concentration of 1-1.5 mg/ml using ultraconcentrators (Vivaspin 15R 30.000 MWCO HY, Sartorius). The proteinconcentration was determined by measuring the optical density (OD) at280 nm, using the molar extinction coefficient calculated on the basisof the amino acid sequence. Purity and molecular weight of theconstructs were analyzed by SDS capillary electrophoresis in thepresence and absence of a reducing agent following the manufacturerinstructions (instrument Caliper LabChipGX, Perkin Elmer). Purifiedproteins were frozen in liquid N₂ and stored at −80° C.

Based on in vitro characterization results selected binders wereconverted into a T-cell bispecific format. In these molecules theFolR1:CD3 binding moieties are arranged in a 2:1 order with the FolR1Fabs being located at the N-terminus. For clones isolated from thestandard Fab library the CD3 binding part was generated as a CrossFab(CH1Cκ crossing) while for the clones from the common light chainlibrary no crossing was necessary. These bispecific molecules wereproduced and purified analogously to the IgGs.

TABLE 6 Yield and monomer content of novel FolR1 binders in IgG and TCBformat, respectively. IgG TCB Yield Monomer Yield Monomer # CloneLibrary [mg/L] [%] [mg/L} [%] 1 11F8 Fab 8.03 96.26 — — 2 14E4 Fab 8.9098.12 — — 3 15B6 CLC 7.72 100.00 — — 4 15E12 CLC 6.19 100.00 — — 5 15H7CLC 8.94 100.00 — — 6 16A3 CLC 0.60 n.d. — — 7 16D5 CLC 36.50 96.96 4.36 97.19 8 16F12 CLC 5.73 97.17 — — 9 18D3 CLC 0.90 n.d. — — 10 19A4 CLC38.32 100.00 37.50  100.00 11 19E5 CLC 46.09 100.00 — — 12 19H3 CLC 7.64100.00 — — 13 20G6 CLC 24.00 100.00 — — 14 20H7 CLC 45.39 100.00 — — 1521A5 CLC 1.38 98.56 47.31   95.08 16 21D1 CLC 5.47 100.00 — — 17 21G8CLC 6.14 97.28 9.27 100.00 18 36F2 Fab 11.22 100.00 18.00  100.00 19 5D9Fab 20.50 100.00 0.93  97.32 20 6B6 Fab 3.83 100.00 4.17  91.53 21 9D11Fab 14.61 100.00 2.63 100.00 CLC: Common light chain

Example 9 2+1 and 1+1 T-Cell Bispecific Formats

Four different T-cell bispecific formats were prepared for one commonlight chain binder (16D5) and three formats for one binder from the Fablibrary (9D11) to compare their killing properties in vitro.

The standard format is the 2+1 inverted format as already described(FolR1:CD3 binding moieties arranged in a 2:1 order with the FolR1 Fabslocated at the N-terminus). In the 2+1 classical format the FolR1:CD3binding moieties are arranged in a 2:1 order with the CD3 Fab beinglocated at the N-terminus. Two monovalent formats were also prepared.The 1+1 head-to-tail has the FolR1:CD3 binding moieties arranged in a1:1 order on the same arm of the molecule with the FolR1 Fab located atthe N-terminus. In the 1+1 classical format the FolR1:CD3 bindingmoieties are present once, each on one arm of the molecule. For the 9D11clone isolated from the standard Fab library the CD3 binding part wasgenerated as a CrossFab (CH1Cκ crossing) while for the 16D5 from thecommon light chain library no crossing was necessary. These bispecificmolecules were produced and purified analogously to the standardinverted T-cell bispecific format.

TABLE 7 Summary of the yield and final monomer content of the differentT-cell bispecific formats. Monomer [%] Construct (SEC) Yield 16D5 FolR1TCB 2 + 1 (inverted) 96% 5.4 mg/L 16D5 FolR1 TCB 2 + 1 (classical) 90%4.6 mg/L 16D5 FolR1 TCB 1 + 1 (head-to- 100% 5.4 mg/L tail) 16D5 FolR1TCB 1 + 1 (classical) 100% 0.7 mg/L 9D11 FolR1 TCB 2 + 1 (inverted) 100%2.6 mg/L 9D11 FolR1 TCB 1 + 1 (head-to- 100% 6.1 mg/L tail) 9D11 FolR1TCB 1 + 1 (classical) 96% 1.3 mg/L Mov19 FolR1 TCB 2 + 1 (inverted) 98%  3 mg/L Mov19 FolR1 TCB 1 + 1 (head-to- 100% 5.2 mg/L tail)

Example 10 Biochemical Characterization of FolR1 Binders by SurfacePlasmon Resonance

Binding of FolR1 binders as IgG or in the T-cell bispecific format todifferent recombinant folate receptors (human FolR1, 2 and 3, murineFolR1 and cynomolgus FolR1; all as Fc fusions) was assessed by surfaceplasmon resonance (SPR). All SPR experiments were performed on a BiacoreT200 at 25° C. with HBS-EP as running buffer (0.01 M HEPES pH 7.4, 0.15M NaCl, 3 mM EDTA, 0.005% Surfactant P20, Biacore, Freiburg/Germany).

Single Injections

First the anti-FolR1 IgGs were analyzed by single injections (Table 1)to characterize their crossreactivity (to human, murine and cyno FolR1)and specificity (to human FolR1, human FolR2, human FolR3). Recombinantbiotinylated monomeric Fc fusions of human, cynomolgus and murine FolateReceptor 1 (FolR1-Fc) or human Folate Receptor 2 and 3 (FolR2-Fc,FolR3-Fc) were directly coupled on a SA chip using the standard couplinginstruction (Biacore, Freiburg/Germany). The immobilization level wasabout 300-400 RU. The IgGs were injected for 60 seconds at aconcentration of 500 nM. IgGs binding to huFolR2 and huFolR3 wererejected for lack of specificity. Most of the binders are onlycrossreactive between human and cyno FolR1, additional crossreactivityto murine FolR1 went most of the time hand in hand with loss ofspecificity.

TABLE 8 Crossreactivity and specificity of 25 new folate receptor 1binders (as IgGs) as well as of two control IgGs (Mov19 andFarletuzumab). Binding to Binding to Binding to Binding to Binding toClone name huFolR1 cyFolR1 muFolR1 huFolR2 huFolR3 Mov19 + + − − −Farletuzumab + + − − − 16A3 + + +/− − − 18D3 + + − − − 19E5 + + + + +19A4 − − + + + 15H7 + + + − − 15B6 + + − − − 16D5 + + − − − 15E12 + ++/− + + 21D1 + + +/− − − 16F12 + + − − − 21A5 + + − − +/− 21G8 + + − + +19H3 − − + − − 20G6 − − + − − 20H7 − − + − − 9D11 + + − − − 5D9 + +− + + 6B6 + + − + + 11F8 + + + + + 36F2 + + + − − 14E4 − − + − − + meansbinding, − means no binding, +/− means weak binding.

Avidity to Folate Receptor 1

The avidity of the interaction between the anti-FolR1 IgGs or T cellbispecifics and the recombinant folate receptors was determined asdescribed below (Table 9).

Recombinant biotinylated monomeric Fc fusions of human, cynomolgus andmurine Folate Receptor 1 (FolR1-Fc) were directly coupled on a SA chipusing the standard coupling instruction (Biacore, Freiburg/Germany). Theimmobilization level was about 300-400 RU. The anti-FolR1 IgGs or T cellbispecifics were passed at a concentration range from 2.1 to 500 nM witha flow of 30 μL/minutes through the flow cells over 180 seconds. Thedissociation was monitored for 600 seconds. Bulk refractive indexdifferences were corrected for by subtracting the response obtained onreference flow cell immobilized with recombinant biotinylated IL2receptor Fc fusion. For the analysis of the interaction of 19H3 IgG andmurine folate receptor 1, folate (Sigma F7876) was added in the HBS-EPrunning buffer at a concentration of 2.3 μM. The binding curvesresulting from the bivalent binding of the IgGs or T cell bispecificswere approximated to a 1:1 Langmuir binding and fitted with that model(which is not correct, but gives an idea of the avidity). The apparentavidity constants for the interactions were derived from the rateconstants of the fitting using the Bia Evaluation software (GEHealthcare).

TABLE 9 Bivalent binding (avidity with apparent KD) of selected FolR1binders as IgGs or as T-cell bispecifics (TCB) on human and cyno FolR1.Apparent Analyte Ligand ka (1/Ms) kd (1/s) KD (M) 16D5 TCB huFolR18.31E+04 3.53E−04 4.24E−09 cyFolR1 1.07E+05 3.70E−04 3.45E−09 9D11 TCBhuFolR1 1.83E+05 9.83E−05 5.36E−10 cyFolR1 2.90E+05 6.80E−05 2.35E−1021A5 TCB huFolR1 2.43E+05 2.64E−04 1.09E−09 cyFolR1 2.96E+05 2.76E−049.32E−10 36F2 IgG huFolR1 2.62E+06 1.51E−02 5.74E−9  cyFolR1 3.02E+061.60E−02 5.31E−9  muFolR1  3.7E+05 6.03E−04 1.63E−9  Mov19 IgG huFolR18.61E+05 1.21E−04  1.4E−10 cyFolR1 1.29E+06 1.39E−04 1.08E−10Farletuzumab huFolR1 1.23E+06   9E−04  7.3E−10 cyFolR1 1.33E+06 8.68E−04 6.5E−10 19H3 IgG muFolR1  7.1E+05  1.1E−03 1.55E−09

1. Affinity to Folate Receptor 1

The affinity of the interaction between the anti-FolR1 IgGs or the Tcell bispecifics and the recombinant folate receptors was determined asdescribed below (Table 10).

For affinity measurement, direct coupling of around 6000-7000 resonanceunits (RU) of the anti-human Fab specific antibody (Fab capture kit, GEHealthcare) was performed on a CM5 chip at pH 5.0 using the standardamine coupling kit (GE Healthcare). Anti-FolR1 IgGs or T cellbispecifics were captured at 20 nM with a flow rate of 10 μl/min for 20or 40 sec, the reference flow cell was left without capture. Dilutionseries (6.17 to 500 nM or 12.35 to 3000 nM) of human or cyno FolateReceptor 1 Fc fusion were passed on all flow cells at 30 μl/min for 120or 240 sec to record the association phase. The dissociation phase wasmonitored for 240 s and triggered by switching from the sample solutionto HBS-EP. The chip surface was regenerated after every cycle using adouble injection of 60 sec 10 mM Glycine-HCl pH 2.1 or pH 1.5. Bulkrefractive index differences were corrected for by subtracting theresponse obtained on the reference flow cell 1. The affinity constantsfor the interactions were derived from the rate constants by fitting toa 1:1 Langmuir binding using the Bia Evaluation software (GEHealthcare).

TABLE 10 Monovalent binding (affinity) of selected FolR1 binders as IgGsor as T-cell bispecifics (TCB) on human and cyno FolR1. Ligand Analyteka (1/Ms) kd (1/s) KD (M) 16D5 TCB huFolR1 1.53E+04 6.88E−04 4.49E−08cyFolR1 1.32E+04 1.59E−03 1.21E−07 9D11 TCB huFolR1 3.69E+04 3.00E−048.13E−09 cyFolR1 3.54E+04 2.06E−04 5.82E−09 21A5 TCB huFolR1 1.79E+04 1.1E−03 6.16E−08 cyFolR1 1.48E+04 2.06E−03  1.4E−07 Mov19 IgG huFolR12.89E+05 1.59E−04  5.5E−10 cyFolR1 2.97E+05 1.93E−04  6.5E−10Farletuzumab huFolR1 4.17E+05 2.30E−02 5.53E−08 cyFolR1 5.53E+053.73E−02 6.73E−08

2. Affinity to CD3

The affinity of the interaction between the anti-FolR1 T cellbispecifics and the recombinant human CD3εδ-Fc was determined asdescribed below (Table 11).

For affinity measurement, direct coupling of around 9000 resonance units(RU) of the anti-human Fab specific antibody (Fab capture kit, GEHealthcare) was performed on a CM5 chip at pH 5.0 using the standardamine coupling kit (GE Healthcare). Anti-FolR1 T cell bispecifics werecaptured at 20 nM with a flow rate of 10 μl/min for 40 sec, thereference flow cell was left without capture. Dilution series (6.17 to500 nM) of human CD3εδ-Fc fusion were passed on all flow cells at 30μl/min for 240 sec to record the association phase. The dissociationphase was monitored for 240 s and triggered by switching from the samplesolution to HBS-EP. The chip surface was regenerated after every cycleusing a double injection of 60 sec 10 mM Glycine-HCl pH 2.1. Bulkrefractive index differences were corrected for by subtracting theresponse obtained on the reference flow cell 1. The affinity constantsfor the interactions were derived from the rate constants by fitting toa 1:1 Langmuir binding using the Bia Evaluation software (GEHealthcare).

TABLE 11 Monovalent binding (affinity) of selected FolR1 T-cellbispecifics (TCB) on human CD3-Fc. Ligand Analyte ka (1/Ms) kd (1/s) KD(M) 16D5 TCB huCD3 4.25E+04 3.46E−03 8.14E−08 21A5 TCB huCD3 3.72E+043.29E−03  8.8E−08

The CD3 binding part is identical for all constructs and the affinity issimilar for the measured T cell bispecifics (KD range between 60 and 90nM).

Example 11 Simultaneous Binding T Cell Bispecifics on Folate Receptor 1and CD3

Simultaneous binding of the anti-FolR1 T cell bispecifics on recombinantFolate Receptor 1 and recombinant human CD3εδ-Fc was determined bysurface plasmon resonance as described below. Recombinant biotinylatedmonomeric Fc fusions of human, cynomolgus and murine Folate Receptor 1(FolR1-Fc) were directly coupled on a SA chip using the standardcoupling instruction (Biacore, Freiburg/Germany). The immobilizationlevel was about 300-400 RU. The anti-FolR1 T cell bispecifics wereinjected for 60 s at 500 nM with a flow of 30 μL/minutes through theflow cells, followed by an injection of hu CDεδ-Fc for 60 s at 500 nM.Bulk refractive index differences were corrected for by subtracting theresponse obtained on reference flow cell immobilized with recombinantbiotinylated IL2 receptor Fc fusion. The four T cell bispecifics tested(16D5 TCB, 21A5 TCB, 51C7 TCB and 45D2 TCB) were able to bindsimultaneously to Folate Receptor 1 and human CD3 as expected.

Example 12 Epitope Binning

For epitope binning, the anti-FolR1 IgGs or T cell bispecifics weredirectly immobilized on a CM5 chip at pH 5.0 using the standard aminecoupling kit (GE Healthcare), with a final response around 700 RU. 500nM huFolR1-Fc was then captured for 60 s, followed by 500 nM of thedifferent binders for 30 s. The surface was regenerated with twoinjections of 10 mM glycine pH 2 for 30 s each. It is assessed if thedifferent binders can bind to huFolR1 captured on immobilized binders(Table 12).

TABLE 12 Epitope characterization of selected FolR1 binders as IgGs oras T-cell bispecifics (TCB) on human FolR1. Analytes in solution On 16D521A5 9D11 36F2 Mov19 huFolR1 TCB TCB TCB IgG IgG FarletuzumabImmobilized 16D5 TCB − − − + + + 21A5 TCB − − − + + + 9D11 TCB Noadditional binding on FolR1 possible once captured on 9D11 36F2 IgGMeasure not possible, huFolR1 dissociates too rapidly Mov19 IgG + + +/−− − − + means binding, − means no binding, +/− means weak binding

Based on these results and additional data with simultaneous binding onimmobilized huFolR1, the binders were separated in three groups. It isnot clear if 9D11 has a separate epitope because it displaces all theother binders. 16D5 and 21A5 seem to be in the same group and Mov19,Farletuzumab (Coney et al., Cancer Res. 1991 Nov. 15; 51(22):6125-32;Kalli et al., Curr Opin Investig Drugs. 2007 December; 8(12):1067-73)and 36F2 in another (Table 13). However, 36F2 binds to a differentepitope than Mov 19 and Farletuzumab as it binds to human, cynomous andmurine FolR1.

TABLE 13 Epitope grouping of selected FolR1 binders as IgGs or as T-cellbispecifics (TCB) on human FolR1 Epitope 1 Epitope 2 Epitope 3 16D5 9D11Mov19 21A5 Farletuzumab 36F2

Example 13 Selection of Binders

FolR1 binders in the IgG formats were screened by surface plasmonresonance (SPR) and by in vitro assay on cells to select the bestcandidates.

The anti-FolR1 IgGs were analyzed by SPR to characterize theircrossreactivity (to human, murine and cynomolgus FolR1) and specificity(to human FolR1, human FolR2, human FolR3). Unspecific binding to humanFolR2 and 3 was considered an exclusion factor. Binding and specificityto human FolR1 was confirmed on cells. Some binders did not bind oncells expressing FolR1 even though they recognized the recombinant humanFolR1 in SPR. Aggregation temperature was determined but was not anexclusion factor because the selected binders were all stable. Selectedbinders were tested in a polyreactivity ELISA to check for unspecificbinding, which led to the exclusion of four binders. This processresulted in an initial selection of three binders: 36F2 (Fab library),9D11 (Fab library) and 16D5 (common light chain). 36F2 dissociatedrapidly from huFolR1 and was, therefore, initially not favored.

Example 14 Specific Binding of Newly Generated FolR1 Binders to HumanFolR1 Positive Tumor Cells

New FolR1 binders were generated via Phage Display using either a Fablibrary or a common light chain library using the CD3 light chain. Theidentified binders were converted into a human IgG1 format and bindingto FolR1 high expressing HeLa cells was addressed. As reference moleculethe human FolR1 binder Mov19 was included. Most of the binders tested inthis assay showed intermediate to good binding to FolR1 with some clonesbinding equally well as Mov19 (see FIG. 2). The clones 16A3, 18D3, 15H7,15B6, 21D1, 14E4 and 16F12 were excluded because binding to FolR1 oncells could not be confirmed by flow cytometry. In a next step theselected clones were tested for specificity to human FolR1 by excludingbinding to the closely related human FolR2. HEK cells were transientlytransfected with either human FolR1 or human FolR2 to addressspecificity. The clones 36F2 and 9D11 derived from the Fab library andthe clones 16D5 and 21A5 derived from the CLC library bind specificallyto human FolR1 and not to human FolR2 (see FIGS. 3A-B). All the othertested clones showed at least some binding to human FolR2 (see FIGS.3A-B). Therefore these clones were excluded from furthercharacterization. In parallel cross-reactivity of the FolR1 clones tocyno FolR1 was addressed by performing binding studies to HEK cellstransiently transfected with cyno FolR1. All tested clones were able tobind cyno FolR1 and the four selected human FoLR1 specific clones 36F2,9D11, 16D5 and 21A5 bind comparably well human and cyno FoLR1 (FIG. 4).Subsequently three human FolR1 specific cyno cross-reactive binders wereconverted into TCB format and tested for induction of T cell killing andT cell activation. These clones were 9D11 from the Fab library and 16D5and 21A5 from the CLC library. As reference molecule Mov19 FolR1 TCB wasincluded in all studies. These FolR1 TCBs were then used to compareinduction of internalization after binding to FolR1 on HeLa cells. Allthree tested clones are internalized upon binding to FolR1 comparable tointernalization upon binding of Mov19 FoLR1 TCB (FIG. 5). 21A5 FolR1 TCBwas discontinued due to signs of polyreactivity.

Example 15 T Cell-Mediated Killing of FolR1-Expressing Tumor TargetCells Induced by FolR1 TCB Antibodies

The FolR1 TCBs were used to determine T cell mediated killing of tumorcells expressing FoLR1. A panel of potential target cell lines was usedto determine FoLR1 binding sites by Qifikit analysis. The used panel oftumor cells contains FolR1 high, intermediate and low expressing tumorcells and a FolR1 negative cell line.

TABLE 14 FolR1 binding sites on tumor cells Cell line Origin FolR1binding sites Hela Cervix adenocarcinoma 2′240′716 Skov3 Ovarianadenocarcinoma 91′510 OVCAR5 Ovarian adenocarcinoma 22′077 HT29Colorectal adenocarcinoma 10′135 MKN45 Gastric adenocarcinoma 54

Binding of the three different FoLR1 TCBs (containing 9D11, 16D5 andMov19 binders) to this panel of tumor cell lines was determined showingthat the FoLR1 TCBs bind specifically to FolR1 expressing tumor cellsand not to a FoLR1 negative tumor cell line. The amount of boundconstruct is proportional to the FolR1 expression level and there isstill good binding of the constructs to the FolR1 low cell line HT-29detectable. In addition there is no binding of the negative control DP47TCB to any of the used cell lines (FIGS. 6A-E).

The intermediate expressing cell line SKOV3 and the low expressing cellline HT-29 were further on used to test T cell mediated killing and Tcell activation using 16D5 TCB and 9D11 TCB; DP47 TCB was included asnegative control. Both cell lines were killed in the presence of alreadyvery low levels of 16D5 TCB and 9D11 TCB and there was no difference inactivity between both TCBs even though 9D11 TCB binds stronger to FolR1than 16D5 TCB. Overall killing of SKOV3 cells was higher compared toHT-29 which reflects the higher expression levels of FolR1 on SKOV3cells (FIGS. 7A-D). In line with this, a strong upregulation of theactivation marker CD25 and CD69 on CD4⁺ T cells and CD8⁺ T cells wasdetected. Activation of T cells was very similar in the presence ofSKOV3 cells and HT-29 cells. The negative control DP47 TCB does notinduce any killing at the used concentrations and there was nosignificant upregulation of CD25 and CD69 on T cells.

TABLE 15 EC50 values of tumor cell killing and T cell activation withSKOV3 cells Killing Killing CD4+ CD4+ CD8+ CD8+ 24 h 48 h CD69+ CD25+CD69+ CD25+ Construct (pM) (pM) (%) (%) (%) (%) 9D11 1.1 0.03 0.51 0.460.019 0.03 FolR1 TCB 16D5 0.7 0.04 0.34 0.33 0.025 0.031 FolR1 TCB

TABLE 16 EC50 values of tumor cell killing and T cell activation withHT-29 cells Killing Killing CD4+ CD4+ CD8+ CD8+ 24 h 48 h CD69+ CD25+CD69+ CD25+ Construct (pM) (pM) (%) (%) (%) (%) 9D11 2.3 0.1 1.22 1.110.071 0.084 FolR1 TCB 16D5 2.8 0.1 0.69 0.62 0.021 0.028 FolR1 TCB

Example 16 Binding to Erythrocytes and T Cell Activation in Whole Blood

To prove that there is no spontaneous activation in the absence of FoLR1expressing tumor cells we tested if there is binding of the FolR1 clonesto erythrocytes which might potentially express FolR1. We could notobserve any specific binding of 9D11 IgG, 16D5 IgG and Mov19 IgG toerythrocytes, as negative control DP47 IgG was included (FIG. 8).

To exclude any further unspecific binding to blood cells or unspecificactivation via FoLR1 TCB, 9D11 TCB, 16D5 TCB and Mov19 TCB were addedinto whole blood and upregulation of CD25 and CD69 on CD4⁺ T cells andCD8⁺ T cells was analyzed by flow cytometry. DP47 TCB was included asnegative control. No activation of T cells with any of the testedconstructs could be observed by analyzing upregulation of CD25 and CD69on CD4⁺ T cells and CD8⁺ T cells (FIG. 9).

Example 17 Removal of the N-Glycosylation Site in 9D11 Light Chain

During analysis of the different FolR1 binders to identify potentialsequence hot spots, at the end of CDR L3 of the clone 9D11 a putativeN-glycosylation site was identified. Usually the consensus motif forN-glycosylation is defined as N-X-S/T-X (where X is not P). The sequenceof CDR L3 (MQASIMNRT) (SEQ ID NO: 61) perfectly matches this consensusmotif having the sequence N-R-T. Since glycosylation might not becompletely reproducible among different production batches this couldhave an impact on FolR1 binding, if the glycosylation in CDR L3contributes to antigen binding. To evaluate if this N-glycosylation siteis important for FolR1 binding, or could be replaced without impairingbinding, different variants of the 9D11 light chain were generated inwhich the N-glycosylation site was exchanged by site specificmutagenesis.

1. Transient Transfection and Production

The four T cell bispecifics were transiently produced in HEK293 EBNAcells using a PEI mediated transfection procedure for the requiredvectors as described below. HEK293 EBNA cells were cultivated insuspension serum free in CD CHO culture medium. For the production in500 ml shake flask 400 million HEK293 EBNA cells were seeded 24 hoursbefore transfection (for alternative scales all amounts were adjustedaccordingly). For transfection cells were centrifuged for 5 min by210×g, supernatant was replaced by pre-warmed 20 ml CD CHO medium.Expression vectors were mixed in 20 ml CD CHO medium to a final amountof 200 μg DNA. After addition of 540 μl PEI solution was vortexed for 15s and subsequently incubated for 10 min at room temperature. Afterwardscells were mixed with the DNA/PEI solution, transferred to a 500 mlshake flask and incubated for 3 hours by 37° C. in an incubator with a5% CO2 atmosphere. After incubation time 160 ml F17 medium was added andcell were cultivated for 24 hours. One day after transfection 1 mMvalporic acid and 7% Feed 1 was added. After 7 days cultivationsupernatant was collected for purification by centrifugation for 15 minat 210×g, the solution is sterile filtered (0.22 μm filter) and sodiumazide in a final concentration of 0.01% w/v was added, and kept at 4° C.After production the supernatants were harvested and the antibodycontaining supernatants were filtered through 0.22 μm sterile filtersand stored at 4° C. until purification.

2. Antibody Purification

All molecules were purified in two steps using standard procedures, suchas protein A affinity purification (Äkta Explorer) and size exclusionchromatography. The supernatant obtained from transient production wasadjusted to pH 8.0 (using 2 M TRIS pH 8.0) and applied to HiTrap PA HP(GE Healthcare, column volume (cv)=5 ml) equilibrated with 8 columnvolumes (cv) buffer A (20 mM sodium phosphate, 20 mM sodium citrate, 0.5M NaCl, 0.01% Tween-20, pH 7.5). After washing with 10 cv of buffer A,the protein was eluted using a pH gradient to buffer B (20 mM sodiumcitrate pH 2.5, 0.5 M NaCl, 0.01% Tween-20) over 20 cv. Fractionscontaining the protein of interest were pooled and the pH of thesolution was gently adjusted to pH 6.0 (using 2 M Tris pH 8.0). Sampleswere concentrated to 1 ml using ultra-concentrators (Vivaspin 15R 30.000MWCO HY, Sartorius) and subsequently applied to a Superdex™ 200 10/300GL (GE Healthcare) equilibrated with 20 mM Histidine, pH 6.0, 140 mMNaCl, 0.01% Tween-20. The aggregate content of eluted fractions wasanalyzed by analytical size exclusion chromatography. Therefore, 30 μlof each fraction was applied to a TSKgel G3000 SW XL analyticalsize-exclusion column (Tosoh) equilibrated in 25 mM K₂HPO₄, 125 mM NaCl,200 mM L-arginine monohydrochloride, 0.02% (w/v) NaN₃, pH 6.7 runningbuffer at 25° C. Fractions containing less than 2% oligomers were pooledand concentrated to final concentration of 1-1.5 mg/ml using ultraconcentrators (Vivaspin 15R 30.000 MWCO HY, Sartorius). The proteinconcentration was determined by measuring the optical density (OD) at280 nm, using the molar extinction coefficient calculated on the basisof the amino acid sequence. Purity and molecular weight of theconstructs were analyzed by SDS capillary electrophoresis in thepresence and absence of a reducing agent following the manufacturerinstructions (instrument Caliper LabChipGX, Perkin Elmer). Purifiedproteins were frozen in liquid N₂ and stored at −80° C.

3. Aggregation Temperature

Stability of the four constructs was tested on an Optim 1000 (Avacta,PALL Corporation) by a gradient heating from 25° to 80° at 0.1° C./min.The temperature at onset of aggregation is recorded.

TABLE 34 Yield, monomer content and aggregation temperature of fourN-glycosylation site knock-out mutant of the 9D11 binder in the 2 + 1inverted T-cell bispecific format. All four mutants behaved similarly tothe wild-type 9D11 binder Yield Monomer Aggregation Clone Mutation[mg/L] [%] temperature 9D11 T102N 1.34 97 56° 9D11 T102A 1.29 100 56°9D11 N100Q 2.5 100 56° 9D11 N100S 2.05 100 56° 9D11 — 2.6 100 57°

The following variants were generated: N100S (N95S); N100Q (N95Q), T102A(T97A) and T102N (T97N) (Kabat numbering indicated in parenthesis) andconverted into the T-cell bispecific format. After transient productionin HEK293 EBNA cells and purification the different variants wereanalyzed for target binding and cell killing activity in comparison tothe original 9D11 clone.

TABLE 17 Primers used for removal of N-glycosylation site in CDR L3 of9D11 (sequences see below) # Amino acid exchange Mutagenesis primer 1N95S GAB-7735 2 N95Q GAB-7734 3 T97A GAB7736 4 T97N GAB-7737

Example 18 Binding and T Cell Mediated Killing with 9D11 a-GlycoVariants

Due to a glycosylation site in the CDRs four different 9D11 variantswere produced with a mutation removing the glycosylation site (Example17). These four variants were tested in comparison to the original 9D11for binding to FolR1 on HeLa cells (FIG. 10) and induction of tumor cellkilling on SKOV3 and HT-29 (FIG. 11A-B, E-F). None of the variantsshowed differences in binding or induction of tumor cell killing. Inparallel unspecific killing of the FolR1 negative cell lines MKN-45 wasaddressed (FIGS. 11C-D). Also, no differences between the variants andthe original binder could be observed. None of the constructs inducedunspecific killing on FoLR1 negative tumor cells.

Example 19 FolR1 Expression on Primary Epithelial Cells

FolR1 is expressed at low levels on primary epithelial cells. Here wewanted to test if these levels are sufficient to induce T cell mediatedkilling in the presence of the FolR1 TCBs. To test this we used primaryhuman bronchial epithelial cells, primary human choroid plexusepithelial cell, primary human renal cortical epithelial cells andprimary human retinal pigment epithelial cells. As positive controleither FolR1 positive SKOV3 cells or HT-29 cells were included. First weverified FolR1 expression on the used primary cells and determined theamount of FolR1 binding sites on these cells. Bronchial epithelialcells, renal cortical epithelial cells and retinal pigment epithelialcells express very low but significant levels of FolR1 compared to thelevels expressed on tumor cells. The choroid plexus epithelial cells donot express significant levels of FolR1.

TABLE 18 FolR1 binding sites on primary epithelial cells Cell lineBinding sites Bronchial epithelium 492 Choroid plexus epithelium 104Renal cortical epithelium 312 Retinal pigment epithelium 822 Skov369′890

The primary epithelial cells that demonstrated FolR1 expression on thesurface were used to address the question if these cells can be killedby T cells in the presence of FoLR1 TCBs. No significant levels ofkilling could be measured but induction of T cell activation in thepresence of retinal pigment epithelial cells, bronchial epithelial cellsand renal cortical cells resulting in upregulation of CD25 and CD69 wasdetected. The strongest activation is seen with retinal pigmentepithelial cells resulting in upregulation of CD25 and CD69 both on CD4⁺T cells and CD8⁺ T cells. In the presence of bronchial epithelial cellslower activation of T cells is induced with upregulation of CD69 on CD4⁺T cells and CD8⁺ T cells but very low upregulation of CD25 only on CD4⁺T cells but not on CD8⁺ T cells. The lowest activation of T cells isobtained in the presence of renal epithelial cells with no upregulationof CD25 on CD4 T⁺ cells and CD8⁺ T cells and CD69 been only upregulatedon CD8⁺ T cells (FIGS. 12A-X).

Example 20 Comparison of Different TCB Formats Containing Either 16D5 or9D11 Binder

To determine if the TCB 2+1 inverted format is the most active formatwith the selected FolR1 binder, different formats containing either 16D5or 9D11 were produced and compared in target cell binding, T cellmediated killing and T cell activation. The 16D5 binder was tested inthe TCB 2+1 inverted (FIG. 1A), TCB 2+1 classical (FIG. 1D), TCB 1+1classical (FIG. 1C) and TCB 1+1 head-to-tail (FIG. 1B) format; the 9D11binder was tested in the TCB 2+1 inverted (FIG. 1A), TCB 1+1 classical(FIG. 1C) and TCB 1+1 head-to-tail (FIG. 1B) format.

All constructs were tested for binding to FolR1 on HeLa cells. Themolecules bivalent for binding to FolR1 bind stronger compared to themonovalent constructs due to avidity. The difference between thebivalent vs. monovalent constructs is more pronounced for 16D5. Thereason might be that due to the lower affinity of 16D5 the avidityeffect for this binder is stronger. Between the two 1+1 TCBs there is nosignificant difference in binding but there is a difference between thetwo 2+1 constructs. The inverted 2+1 construct binds stronger to FolR1than the classical 2+1 construct. This indicates that in the classical2+1 construct the binding to FoLR1 is influenced by the presence of theCD3 Fab whereas in the inverted construct binding is less influenced.

By testing T cell mediated killing with these constructs we could showthat stronger binding of the 2+1 inverted TCB in converted into strongertumor cell killing and T cell activation compared to the 2+1 classicalTCB. The 16D5 FoLR1 TCB 2+1 classical is only a little bit more activethan the respective 1+1 head-to-tail construct. The 1+1 head-to-tailconstruct is significantly more active than the 1+1 classical construct.This does not reflect the situation seen in binding and might be due tobetter crosslinking with the head-to-tail construct. Overall tumor cellkilling and T cell activation is comparable with all tested constructs,the differences in potency seen with the differences are only in termsof EC50 values. In general it can be concluded that the FolR1 TCB 2+1inverted independent of the used binder is the preferred format toinduce T cell mediated tumor cell killing and T cell activation (seeFIG. 13A-C and FIG. 14A-C).

TABLE 19 EC50 values of target cell binding and T cell mediated killingwith different TCB formats Killing 24 h Killing 48 h Construct BindingEC50 (nM) (pM) (pM) 16D5 FolR1 TCB 11.03 1.43 0.18 2 + 1 inverted 16D5FolR1 TCB 17.07 5.60 2.18 2 + 1 classical 16D5 FolR1 TCB 107.3 n.d. n.d.1 + 1 classical 16D5 FoLR1 TCB 102.6 26.24 6.06 1 + 1 head-to-tail 9D11FoLR1 TCB 17.52 0.74 0.14 2 + 1 inverted 9D11 FoLR1 TCB 38.57 20.92 n.d.1 + 1 classical 9D11 FoLR1 TCB 44.20 4.73 n.d. 1 + 1 head-to-tail

TABLE 20 EC50 values of T cell activation in the presence of SKOV3 cellswith different TCB formats CD4+CD25+ CD4+CD69+ CD8+CD25+ CD8+CD69+Construct (%) (%) (%) (%) 16D5 1.96 0.33 2.10 n.d. FolR1 TCB 2 + 1inverted 16D5 13.83 3.67 12.88 4.47 FolR1 TCB 2 + 1 classical 16D5 38.54n.d. n.d. n.d. FolR1 TCB 1 + 1 classical 16D5 FoLR1 17.14 7.47 25.15n.d. TCB 1 + 1 head-to-tail 9D11 1.41 0.27 1.24 0.35 FoLR1 TCB 2 + 1inverted 9D11 34.01 n.d. 34.39 7.40 FoLR1 TCB 1 + 1 classical 9D11 FoLR13.73 2.47 4.98 2.89 TCB 1 + 1 head-to-tail

Example 21 Tumor Cell Lines and Primary Cells

HeLa cells (CCL-2) were obtained from ATCC and cultured in DMEM with 10%FCS and 2 mM Glutamine, SKOV3 (HTB-77) were obtained from ATCC andcultured in RPMI with 10% FCS and 2 mM Glutamine, OVCAR5 were obtainedfrom NCI and cultured in RPMI with 10% FCS and 2 mM Glutamine, HT-29(ACC-299) were obtained from DSMZ and cultured in McCoy's 5A medium with10% FCS and 2 mM Glutamine, MKN-45 (ACC-409) were obtained from DSMZ andcultured in RPMI with 10% FCS and 2 mM Glutamine.

All tested primary epithelial cells were obtained from ScienCellResearch Laboratories. Human Bronchial Epithelium Cells (HBEpiC, CatalogNumber 3210 were cultured in Bronchial Epithelial Cell Medium (BEpiCM,Cat. No. 3211, ScienCell). Human Colonic Epithelial Cells (HCoEpiC),Catalog Number 2950 were cultured in Colonic Epithelial Cell Medium(CoEpiCM, Cat. No. 2951, ScienCell). Human Retinal Pigment EpithelialCells (HRPEpiC), Catalog Number 6540 were cultured in Epithelial CellMedium (EpiCM, Cat. No. 4101, ScienCell). Human Renal CorticalEpithelial Cells (HRCEpiC), Catalog Number 4110, were cultured inEpithelial Cell Medium (EpiCM, Cat. No. 4101, ScienCell). Human ChoroidPlexus Epithelial Cells (HCPEpiC), Catalog Number 1310 were cultured inEpithelial Cell Medium (EpiCM, Cat. No. 4101, ScienCell).

Example 22 Target Binding by Flow Cytometry

Target cells as indicated were harvested with Cell Dissociation Buffer,washed with PBS and resuspended in FACS buffer. The antibody stainingwas performed in a 96well round bottom plate. Therefore 200′000 cellswere seeded per well. The plate was centrifuged for 4 min at 400 g andthe supernatant was removed. The test antibodies were diluted in FACSbuffer and 20 μl of the antibody solution were added to the cells for 30min at 4° C. To remove unbound antibody the cells were washed twice withFACS buffer before addition of the diluted secondary antibody (FITCconjugated AffiniPure F(ab′)2 fragment goat anti-human IgG, FcgFragment, Jackson ImmunoResearch #109-096-098 or PE-conjugatedAffiniPure F(ab′)2 Fragment goat anti-human IgG Fcg Fragment Specific,Jackson ImmunoResearch #109-116-170. After 30 min incubation on 4° C.unbound secondary antibody was washed away. Before measurement the cellswere resuspended in 200 μl FACS buffer and analyzed by flow cytometryusing BD Canto II or BD Fortessa.

Example 23 Internalization

The cells were harvested and the viability was determined. The cellswere re-suspended in fresh cold medium at 2 Mio cells per ml and thecell suspension was transferred in a 15 ml falcon tube for eachantibody. The antibodies that should be tested for internalization wereadded with a final concentration of 20 μg per ml to the cells. The tubeswere incubated for 45 min in the cold room on a shaker. After incubationthe cells were washed three times with cold PBS to remove unboundantibodies. 0.2 Mio cells per well were transfer to the FACS plate astime point zero. The labeled cells were re-suspended in warm medium andincubated at 37° C. At the indicated time-points 0.2 Mio cells per wellwere transferred in cold PBS, washed in plated on the FACS plate. Todetect the constructs that remain on the surface the cells were stainedwith PE-labeled anti-human Fc secondary antibody. Therefore 20 μl of thediluted antibody were added per well and the plate was incubated for 30min at 4° C. Then the cells were washed twice to remove unboundantibodies and then fixed with 1% PFA to prevent any furtherinternalization. The fluorescence was measured using BD FACS CantoII.

Example 24 QIFIKIT® Analysis

QIFIKIT® contains a series of beads, 10 μm in diameter and coated withdifferent, but well-defined quantities of mouse Mab molecules(high-affinity anti-human CD5, Clone CRIS-1, isotype IgG2a). The beadsmimic cells with different antigen densities which have been labeledwith a primary mouse Mab, isotype IgG. Briefly, cells were labeled withprimary mouse monoclonal antibody directed against the antigen ofinterest. In a separate test well, cells were labeled with irrelevantmouse monoclonal antibody (isotype control). Then, cells, Set-Up Beadsand Calibration Beads were labeled with a fluorescein-conjugatedanti-mouse secondary antibody included in the kit. The primary antibodyused for labeling of the cells has to be used at saturatingconcentration. The primary antibody may be of any mouse IgG isotype.Under these conditions, the number of bound primary antibody moleculescorresponds to the number of antigenic sites present on the cellsurface. The secondary antibody is also used at saturatingconcentration. Consequently, the fluorescence is correlated with thenumber of bound primary antibody molecules on the cells and on thebeads.

Example 25 T Cell Mediated Tumor Cell Killing and T Cell Activation

Target cells were harvested with Trypsin/EDTA, counted and viability waschecked. The cells were resuspended in their respective medium with afinal concentration of 300′000 cells per ml. Then 100 μl of the targetcell suspension was transferred into each well of a 96-flat bottomplate. The plate was incubated overnight at 37° C. in the incubator toallow adherence of the cells to the plate. On the next day PBMCs wereisolated from whole blood from healthy donors. The blood was diluted 2:1with PBS and overlayed on 15 ml Histopaque-1077 (#10771, Sigma-Aldrich)in Leucosep tubes and centrifuged for 30 min at 450 g without break.After centrifugation the band containing the cells was collected with a10 ml pipette and transferred into 50 ml tubes. The tubes were filled upwith PBS until 50 ml and centrifuged (400 g, 10 min, room temperature).The supernatant was removed and the pellet resuspended in PBS. Aftercentrifugation (300 g, 10 min, room temperature), supernatants werediscarded, 2 tubes were pooled and the washing step was repeated (thistime centrifugation 350×g, 10 min, room temperature). Afterwards thecells were resuspended and the pellets pooled in 50 ml PBS for cellcounting. After counting cells were centrifuged (350 g, 10 min, roomtemperature) and resuspended at 6 Mio cells per ml in RPMI with 2% FCSand 2 nM Glutamine. Medium was removed from plated target cells and thetest antibodies diluted in RPMI with 2% FCS and 2 nM Glutamine wereadded as well as. 300′000 cells of the effector cell solution weretransferred to each well resulting in a E:T ratio of 10:1. To determinethe maximal release target cells were lysed with Triton X-100. LDHrelease was determined after 24 h and 48 h using Cytotoxicity DetectionKit (#1644793, Roche Applied Science). Activation marker upregulation onT cells after tumor cell killing was measured by flow cytometry. BrieflyPBMCs were harvested, transferred into a 96 well round bottom plate andstained with CD4 PE-Cy7 (#3557852, BD Bioscience), CD8 FITC (#555634, BDBioscience), CD25 APC (#555434, BD Bioscience), CD69 PE (#310906,BioLegend) antibodies diluted in FACS buffer. After 30 min incubation at4° C. the cells were washed twice with FACS buffer. Before measuring thefluorescence using BD Canto II the cells were resuspended in 200 μl FACSbuffer.

Example 26 T Cell Activation in Whole Blood

280 μl of fresh blood were added into a 96 well conical deep well plate.Then 20 μl of the diluted TCBs were added to the blood and mixed well byshaking the plate. After 24 h incubation at 37° C. in an incubator theblood was mixed and 35 μl were transferred to a 96well round bottomplate. Then 20 μl of the antibody staining mix were added consisting ofCD4 PE-Cy7 (#3557852, BD Bioscience), CD8 FITC (#555634, BD Bioscience),CD25 APC (#555434, BD Bioscience), CD69 PE (#310906, BioLegend) and CD45V500 (#560777, BD Horizon) and incubated for 15 min in the dark at roomtemperature. Before measuring 200 μl of the freshly prepared BD FACSlysing solution (#349202, BD FCAS) was added to the blood. After 15 minincubation at room temperature the cells were measured with BD Fortessa.

Example 27 SDPK (Single Dose Pharmacokinetics) Study of Humanized FOLR1TCB (Clone 16D5) in Immunodeficient NOD/Shi-Scid/IL-2Rγ null (NOG) Mice

Female NOD/Shi-scid/IL-2Rγ null (NOG) mice, age 6-7 weeks at start ofthe experiment (bred at Taconic, Denmark) were maintained underspecific-pathogen-free condition with daily cycles of 12 h light/12 hdarkness according to committed guidelines (GV-Solas; Felasa; TierschG).The experimental study protocol was reviewed and approved by localgovernment (P 2011/128). After arrival, animals were maintained for oneweek to get accustomed to the new environment and for observation.Continuous health monitoring was carried out on a regular basis.

Mice were injected i.v. with 10/1/0.1 μg/mouse of the FOLR1 TCB whereas3 mice were bled per group and time point. All mice were injected with atotal volume of 200 μl of the appropriate solution. To obtain the properamount of the FOLR1 TCB per 200 the stock solutions were diluted withPBS when necessary. Serum samples were collected 5 min, 1 h, 3 h, 8 h,24 h, 48 h, 72 h, 96 h and 168 h after therapy injection.

FIG. 15 shows that the 16D5 FOLR1 TCB shows typical and doseproportional IgG-like PK properties in NOG mice with slow clearance.

TABLE 21 Experimental conditions. Formulation Concentration CompoundDose buffer (mg/mL) FOLR1 TCB 10 μg 20 mM Histidine, 5.43 (16D5)(corresponding 140 mM NaCl, (=stock solution) to ca. 0.5 mg/kg) pH 6.0FOLR1 TCB 1 μg 20 mM Histidine, 5.43 (16D5) (corresponding 140 mM NaCl,(=stock solution) to ca. 0.05 mg/kg) pH 6.0 FOLR1 TCB 0.1 μg 20 mMHistidine, 5.43 (16D5) (corresponding 140 mM NaCl, (=stock solution) toca. 0.005 mg/kg) pH 6.0

Example 28

In Vivo Efficacy of FOLR1 TCB (Clone 16D5) after Human PBMC Transfer inSkov3-Bearing NOG Mice

The FOLR1 TCB was tested in the human ovarian carcinoma cell line Skov3,injected s.c. into PBMC engrafted NOG mice.

The Skov3 ovarian carcinoma cells were obtained from ATCC (HTB-77). Thetumor cell line was cultured in RPMI containing 10% FCS (Gibco) at 37°C. in a water-saturated atmosphere at 5% CO₂. Passage 35 was used fortransplantation, at a viability >95%. 5×10⁶ cells per animal wereinjected s.c. into the right flank of the animals in a total of 100 μlof RPMI cell culture medium (Gibco).

Female NOD/Shi-scid/IL-2Rγ null (NOG) mice, age 6-7 weeks at start ofthe experiment (bred at Taconic, Denmark) were maintained underspecific-pathogen-free condition with daily cycles of 12 h light/12 hdarkness according to committed guidelines (GV-Solas; Felasa; TierschG).The experimental study protocol was reviewed and approved by localgovernment (P 2011/128). After arrival, animals were maintained for oneweek to get accustomed to the new environment and for observation.Continuous health monitoring was carried out on a regular basis.

According to the protocol (FIG. 16), mice were injected s.c. on studyday 0 with 5×10⁶ of the Skov3. At study day 21, human PBMC of a healthydonor were isolated via the Ficoll method and 10×10⁶ cells were injectedi.p. into the tumor-bearing mice. Two days after, mice were randomizedand equally distributed in five treatment groups (n=12) followed by i.v.injection with either 10/1/0.1 μg/mouse of the FOLR1 TCB or 10 μg/mouseof the DP47 control TCB once weekly for three weeks. All mice wereinjected i.v. with 200 μl of the appropriate solution. The mice in thevehicle group were injected with PBS. To obtain the proper amount of TCBper 200 the stock solutions were diluted with PBS when necessary. Tumorgrowth was measured once weekly using a caliper (FIG. 17) and tumorvolume was calculated as followed:

T _(v): (W ²/2)×L (W:Width,L:Length)

The once weekly injection of the FOLR1 TCB resulted in a dose-dependentanti-tumoral effect. Whereas a dose of 10 μg/mouse and 1 μg/mouseinduced tumor shrinkage and 0.1 μg/mouse a tumor stasis (FIG. 17, Table22). Maximal tumor shrinkage was achieved at a dose of 10 μg/mouse ascompared to a non-targeted control DP47 TCB.

TABLE 22 In vivo efficacy. Tumor growth Compound Dose inhibition DP47TCB 10 μg 7% control TCB (corresponding to ca. 0.5 mg/kg) FOLR1 TCB 10μg 90% (16D5) (corresponding to ca. 0.5 mg/kg) FOLR1 TCB 1 μg 74% (16D5)(corresponding to ca. 0.05 mg/kg) FOLR1 TCB 0.1 μg 56% (16D5)(corresponding to ca. 0.005 mg/kg)

For PD read-outs, three mice per treatment group were sacrificed atstudy day 32, tumors were removed and single cell suspensions wereprepared through an enzymatic digestion with Collagenase V, Dispase IIand DNAse for subsequent FACS-analysis (FIGS. 19 and 20). Single cellswhere either used directly for staining of extracellular antigens andactivation markers or were re-stimulated using 5 ng/ml PMA and 500 ng/mlIonomycin in the presence of a protein transport inhibitor Monensin for5 h in normal culture medium. After re-stimulation, cells were stainedfor surface antigens, followed by a fixation and permeabilization step.Fix samples were then stained intracellulary for TNF-α, IFN-γ, IL-10 andIL-2 and analyzed by flow cytometry. Same procedure was used for thedegranulation of cells, but an anti-CD107a antibody was added during therestimulation period and fixed samples were staining for intracellularperforin and granzyme-B contents. The FACS analysis revealedstatistically higher number of infiltrating CD4⁺ and CD8⁺ T-cells in thetumor tissue upon treatment with FOLR1 TCB compared to vehicle anduntargeted control TCB. Furthermore, higher numbers of TNF-α, IFN-γ andIL-2 producing as well as perforin⁺/granzym-B⁺ CD4⁺ and CD8⁺ T-cellswere detected in FOLR1 TCB treated tumors. Tumor infiltrating T-cellstreated with FOLR1 TCB also showed higher degranulation rates comparedto control groups.

At study termination day 38, all animals were sacrificed; tumors wereremoved and weight (FIG. 18). The weight of the tumors treated with 10and 1 μg/mouse of the FOLR1 TCB showed a statistically significantdifference compared to the control groups.

TABLE 23 Experimental conditions. Concentration Compound DoseFormulation buffer (mg/mL) PBS FOLR1 TCB 10 μg 20 mM Histidine, 3.88(16D5) 140 mM NaCl, (=stock solution) pH 6.0 FOLR1 TCB  1 μg 20 mMHistidine, 3.88 (16D5) 140 mM NaCl, (=stock solution) pH 6.0 FOLR1 TCB0.1 μg  20 mM Histidine, 3.88 (16D5) 140 mM NaCl, (=stock solution) pH6.0 DP47 TCB 10 μg 20 mM Histidine, 4.35 140 mM NaCl, (=stock solution)pH 6.0

Example 29 Generation of a Bispecific FolR1/CD3-Kappa-Lambda Antibody

To generate a bispecific antibody (monovalent for each antigen) thatsimultaneously can bind to human CD3 and human folate receptor alpha(FolR1) without using any hetero-dimerization approach (e.g.knob-into-hole technology), a combination of a common light chainlibrary with the so-called CrossMab technology was applied: The variableregion of the humanized CD3 binder (CH2527_VL7_46/13) was fused to theCH1 domain of a standard human IgG1 antibody to form the VLVH crossedmolecule (fused to Fc) which is common for both specificities. Togenerate the crossed counterparts (VHCL), a CD3 specific variable heavychain domain (CH2527_VH_23/12) was fused to a constant human λ lightchain whereas a variable heavy chain domain specific for human FolR1(clone 16D5, isolated from common light chain library) was fused to aconstant human κ light chain. This enables the purification of thedesired bispecific antibody by applying subsequent purification stepswith KappaSelect and LambdaFab Select columns (GE Healthcare) to removeundesired homodimeric antibodies.

All antibody expression vectors were generated using standardrecombinant DNA technology as described in Sambrook, J. et al.,Molecular cloning: A laboratory manual; Cold Spring Harbor LaboratoryPress, Cold Spring Harbor, N.Y., 1989. Molecular biological reagentswere used according the manufacturer's recommendations. Genes or genefragments were either amplified by polymerase chain reaction (PCR) orgenerated from synthetic oligonucleotides at Geneart AG (Regensburg,Germany) by automated gene synthesis. PCR-amplified or subcloned DNAfragments were confirmed by DNA sequencing (Synergene GmbH,Switzerland). Plasmid DNA was transformed into and amplified in suitableE. coli host strains for preparation of transfection-grade plasmid DNAusing standard Maxiprep kits (Qiagen). For production of the bispecificmolecules HEK293 EBNA cells were transfected with plasmids encoding therespective genes using a standard polyethlenimine (PEI) based method.The used plasmid ratio of the three expression vectors was 1:1:1.Transfected cells were cultivated for 7 days before supernatants wereharvested for purification. The bispecific FolR1/CD3-kappa-lambdaantibodies were produced and purified as follows.

1. Transient Transfection and Production

The kappa-lambda bispecific antibody was transiently produced in HEK293EBNA cells using a PEI mediated transfection procedure for the requiredvectors as described below. HEK293 EBNA cells were cultivated insuspension serum free in CD CHO culture medium. For the production in500 ml shake flask 400 million HEK293 EBNA cells were seeded 24 hoursbefore transfection (for alternative scales all amounts were adjustedaccordingly). For transfection cells were centrifuged for 5 min by210×g, supernatant is replaced by pre-warmed 20 ml CD CHO medium.Expression vectors were mixed in 20 ml CD CHO medium to a final amountof 200 μg DNA. After addition of 540 μl PEI solution is vortexed for 15s and subsequently incubated for 10 min at room temperature. Afterwardscells were mixed with the DNA/PEI solution, transferred to a 500 mlshake flask and incubated for 3 hours by 37° C. in an incubator with a5% CO2 atmosphere. After incubation time 160 ml F17 medium was added andcell were cultivated for 24 hours. One day after transfection 1 mMvalporic acid and 7% Feed 1 was added. After 7 days cultivationsupernatant was collected for purification by centrifugation for 15 minat 210×g, the solution is sterile filtered (0.22 μm filter) and sodiumazide in a final concentration of 0.01% w/v was added, and kept at 4° C.

2. Purification

The kappa-lambda bispecific antibody was purified in three steps, usingan affinity step specific for kappa light chains, followed by anaffinity step specific for lambda light chains and finally by a sizeexclusion chromatography step for removal of aggregates. The supernatantobtained from transient production was adjusted to pH 8.0 (using 2 MTRIS pH 8.0) and applied to Capture Select kappa affinity matrix, orHiTrap KappaSelect, GE Healthcare, column volume (cv)=1 ml, equilibratedwith 5 column volumes (cv) buffer A (50 mM Tris, 100 mM glycine, 150 mMNaCl, pH 8.0). After washing with 15 cv of buffer A, the protein waseluted using a pH gradient to buffer B (50 mM Tris, 100 mM glycine, 150mM NaCl, pH 2.0) over 25 cv. Fractions containing the protein ofinterest were pooled and the pH of the solution was adjusted to pH 8.0(using 2 M Tris pH 8.0). The neutralized pooled fractions were appliedto Capture Select lambda affinity matrix (now: HiTrap LambdaFab Select,GE Healthcare, column volume (cv)=1 ml) equilibrated with 5 columnvolumes (cv) buffer A (50 mM Tris, 100 mM glycine, 150 mM NaCl, pH 8.0).After washing with 15 cv of buffer A, the protein was eluted using a pHgradient to buffer B (50 mM Tris, 100 mM glycine, 150 mM NaCl, pH 2.0)over 25 cv. Fractions containing the protein of interest were pooled andthe pH of the solution was adjusted to pH 8.0 (using 2 M Tris pH 8.0).This solution was concentrated using ultra-concentrators (Vivaspin 15R30.000 MWCO HY, Sartorius) and subsequently applied to a Superdex™ 20010/300 GL (GE Healthcare) equilibrated with 20 mM Histidine, pH 6.0, 140mM NaCl, 0.01% Tween-20. The pooled fractions after size exclusion wereagain concentrated using ultra-concentrators (Vivaspin 15R 30.000 MWCOHY, Sartorius).

The protein concentration was determined by measuring the opticaldensity (OD) at 280 nm, using the molar extinction coefficientcalculated on the basis of the amino acid sequence. Purity and molecularweight of the constructs were analyzed by SDS capillary electrophoresisin the presence and absence of a reducing agent following themanufacturer instructions (instrument Caliper LabChipGX, Perkin Elmer).Only small amounts of protein could be purified with a final yield of0.17 mg/L.

Example 30 T Cell Mediated Killing with BispecificFolR1/CD3-Kappa-Lambda Antibody

Activity of kappa lambda FolR1 TCB was tested on SKOV3 cells in thepresence of freshly isolated PBMCs. As negative control DP47 TCB wasincluded. T cell mediated killing of SKOV3 cells was determined after 24h and 48 h by LDH release. After 48 h the T cells were harvested andCD69 and CD25 upregulation on CD4 T cells and CD8 T cells was measuredby flow cytometry.

The kappa lambda FolR1 construct induces killing of SKOV3 cells in aconcentration dependent manner which is accompanied by CD69 and CD25upregulation both on CD4 T cells and on CD8 T cells.

SKOV3 cells were incubated with PBMCs in the presence of either kappalambda FoLR1 TCB or DP47 TCB. After 24 h and 48 h killing of tumor cellswas determined by measuring LDH release (FIG. 21). SKOV3 cells wereincubated with PBMCs in the presence of either kappa lambda FoLR1 TCB orDP47 TCB. After 48 h CD25 and CD69 upregulation on CD4 T cells and CD8 Tcells was measured by flow cytometry (FIG. 22).

Example 31 Biochemical Characterization of 16D5 and 36F2 FolR1 Bindersby Surface Plasmon Resonance

Binding of anti-FolR1 16D5 in different monovalent or bivalent T-cellbispecific formats and of anti-FolR1 36F2 as IgG or as T-cell bispecificto recombinant human, cynomolgus and murine folate receptor 1 (all as Fcfusions) was assessed by surface plasmon resonance (SPR). All SPRexperiments were performed on a Biacore T200 at 25° C. with HBS-EP asrunning buffer (0.01 M HEPES pH 7.4, 0.15 M NaCl, 3 mM EDTA, 0.005%Surfactant P20, Biacore, GE Healthcare).

1. Molecules Tested

The molecules used for affinity and avidity determination are describedin Table 24.

TABLE 24 Name and description of the 6 constructs used in SPR analysisName Description 16D5 TCB 2 + 1 T-cell bispecific, inverted format(common light chain) 16D5 TCB classical 2 + 1 T-cell bispecific,classical format (common light chain) 16D5 TCB 1 + 1 1 + 1 T-cellbispecific (common light chain) 16D5 TCB 1 + 1 HT 1 + 1 T-cellbispecific head-to- tail (common light chain) 36F2 IgG Human IgG1 withP329G LALA 36F2 TCB 2 + 1 T-cell bispecific, inverted format, crossfab

2. Avidity to Folate Receptor 1

The avidity of the interaction between the anti-FolR1 IgG or T cellbispecifics and the recombinant folate receptors was determined asdescribed below (Table 25).

Recombinant biotinylated monomeric Fc fusions of human, cynomolgus andmurine Folate Receptor 1 (FolR1-Fc) were directly coupled on a SA chipusing the standard coupling instruction (Biacore, GE Healthcare). Theimmobilization level was about 300-400 RU. The anti-FolR1 IgGs or T cellbispecifics were passed at a concentration range from 3.7 to 900 nM witha flow of 30 μL/minutes through the flow cells over 180 seconds. Thedissociation was monitored for 240 or 600 seconds. The chip surface wasregenerated after every cycle using a double injection of 30 sec 10 mMGlycine-HCl pH 2. Bulk refractive index differences were corrected forby subtracting the response obtained on reference flow cell immobilizedwith recombinant biotinylated murine CD134 Fc fusion. The binding curvesresulting from the bivalent binding of the IgG or T cell bispecificswere approximated to a 1:1 Langmuir binding (even though it is a 1:2binding) and fitted with that model to get an apparent KD representingthe avidity of the bivalent binding. The apparent avidity constants forthe interactions were derived from the rate constants of the fittingusing the Bia Evaluation software (GE Healthcare). For the 1+1 T cellbispecifics format the interaction is a real 1:1 and the KD representsaffinity since there is only one FolR1 binder in this construct.

TABLE 25 Bivalent binding (avidity with apparent KD) of anti-FolR1 16D5and 36F2 as IgG or as T-cell bispecifics (TCB) on human, cyno and murineFolR1. Apparent Analyte Ligand ka (1/Ms) kd (1/s) KD 36F2 IgG huFolR12.07E+06  1.3E−02 6 nM cyFolR1 2.78E+06 1.75E−02 6 nM muFolR1 4.28E+058.23E−04 2 nM 36F2 TCB huFolR1 2.45E+06 9.120E−03  4 nM cyFolR1 4.31E+061.45E−02 3 nM muFolR1 6.97E+05 9.51E−04 1 nM 16D5 TCB huFolR1 1.57E+053.92E−04 3 nM cyFolR1 2.01E+05 3.81E−04 2 nM 16D5 TCB classical huFolR12.04E+05 1.84E−04 0.9 nM   cyFolR1 2.50E+05 3.05E−04 1 nM 16D5 TCB 1 + 1HT huFolR1 5.00E+04 2.25E−03 45 nM  cyFolR1 5.75E+04 4.10E−03 70 nM 16D5 TCB 1 + 1 huFolR1 3.65E+04 2.04E−03 56 nM  cyFolR1 4.09E+043.60E−03 90 nM 

3. Affinity to Folate Receptor 1

The affinity of the interaction between the anti-FolR1 IgG or T cellbispecifics and the recombinant folate receptors was determined asdescribed below (Table 26).

For affinity measurement, direct coupling of around 12000 resonanceunits (RU) of the anti-human Fab specific antibody (Fab capture kit, GEHealthcare) was performed on a CM5 chip at pH 5.0 using the standardamine coupling kit (GE Healthcare). Anti-FolR1 IgG or T cell bispecificswere captured at 20 nM with a flow rate of 10 μl/min for 40 sec, thereference flow cell was left without capture. Dilution series (12.3 to3000 nM) of human, cyno or murine Folate Receptor 1 Fc fusion werepassed on all flow cells at 30 μl/min for 240 sec to record theassociation phase. The dissociation phase was monitored for 300 s andtriggered by switching from the sample solution to HBS-EP. The chipsurface was regenerated after every cycle using a double injection of 60sec 10 mM Glycine-HCl pH 1.5. Bulk refractive index differences werecorrected for by subtracting the response obtained on the reference flowcell 1. The affinity constants for the interactions were derived fromthe rate constants by fitting to a 1:1 Langmuir binding using the BiaEvaluation software (GE Healthcare).

TABLE 26 Monovalent binding (affinity) of anti-FolR1 16D5 and 36F2 asIgG or as T-cell bispecifics (TCB) on human, cyno and murine FolR1.Analyte Ligand ka (1/Ms) kd (1/s) KD 36F2 IgG huFolR1 9.10E+04 6.65E−02730 nM cyFolR1 1.02E+05 5.78E−02 570 nM muFolR1 8.32E+04 1.78E−02 210 nM36F2 TCB huFolR1 5.94E+04 6.13E−02 1000 nM  cyFolR1 6.29E+04 5.42E−02860 nM muFolR1 5.68E+04 1.75E−02 300 nM 16D5 TCB huFolR1 2.23E+047.33E−04  33 nM cyFolR1 1.57E+04 1.60E−03 100 nM 16D5 TCB classicalhuFolR1 1.03E+04 7.59E−04  74 nM cyFolR1 9.18E+03 1.61E−03 175 nM 16D5TCB 1 + 1 HT huFolR1 2.05E+04 7.08E−04  35 nM cyFolR1 1.67E+04 1.53E−03 92 nM 16D5 TCB 1 + 1 huFolR1 1.43E+04 9.91E−04  69 nM cyFolR1 1.20E+041.80E−03 150 nM

The affinity (monovalent binding) to human and cyno FolR1-Fc of 36F2 TCBis similar and around 1000 nM for both, whereas the affinity to murineFolR1-Fc is slightly better and around 300 nM. The 36F2 can be used inmurine and primate models, there is no need for a surrogate.

The avidity (apparent KD) of 36F2 TCB to human FolR1 is around 30 timeslower than the affinity of the 16D5 TCB to human FolR1. In the bivalentformat, 36F2 TCB is in the low nanomolar range, whereas 16D5 TCB is inthe low picomolar range (1000 fold difference).

FolR1 is expressed on tumor cells overexpressed, at intermittent andhigh levels, on the surface of cancer cells in a spectrum of epithelialmalignancies, including ovarian, breast, renal, colorectal, lung andother solid cancers and is also expressed on the apical surface of alimited subset of polarized epithelial cells in normal tissue. Thesenon-tumorous, normal cells express FolR1 only at low levels, andinclude, e.g., bronchiolal epithelial cells on alveolar surface, renalcortical luminal border of tubular cells, retinal pigment epithelium(basolateral membrane) and choroid plexus. 16D5 TCB binds to normaltissues cells expressing low amounts of FolR1 which results in their Tcell mediated killing. This might, at least in part, account for limitedtolerance observed at 10 μg/kg in cynomolgus monkeys. The inventorswanted to determine if lowering the affinity of the T cell bispecificmolecule could increase the differentiation between high and low targetdensity tissues and, thereby, lower toxicity by making use of bivalentbinding and avidity. Low affinity binders are ordinarily not selected assuitable candidates for further analysis because low affinity is oftenassociated with low potency and efficacy. Nevertheless, the low affinityFolR1 binder 36F2 was developed in several formats and characterized forits biological properties. For the 36F2 used in the bivalent T cellbispecific format the avidity effect (difference between monovalent andbivalent binding) is around 250 fold (1000 nM versus 4 nM). At lowtarget density the affinity defined the interaction and with 1000 nM ledto a low potency of the TCB. However, at high target density themolecule's avidity comes into play and with 4 nM led to a high activityof the TCB (see Example 32).

In an alternatively approach, the inventors generated monovalent formatsof 16D5 and low affinity variant of 16D5 (affinity about 10-40 nM) in abivalent format. The 16D5 binder used in a monovalent format (1+1) hasan affinity of about 50 nM. The differentiation between high and lowtarget density tissues can be better achieved by taking advantage of theavidity effect.

Example 32 T-Cell Killing of SKov-3 Cells Induced by 36F2 TCB, Mov19 TCBand 21A5 TCB

T-cell killing mediated by 36F2 TCB, Mov19 TCB and 21A5 TCB was assessedon SKov-3 cells (medium FolR1). Human PBMCs were used as effectors andthe killing was detected at 24 h and 48 h of incubation with thebispecific antibodies. Briefly, target cells were harvested withTrypsin/EDTA, washed, and plated at density of 25 000 cells/well usingflat-bottom 96-well plates. Cells were left to adhere overnight.Peripheral blood mononuclear cells (PBMCs) were prepared by Histopaquedensity centrifugation of enriched lymphocyte preparations (buffy coats)obtained from healthy human donors. Fresh blood was diluted with sterilePBS and layered over Histopaque gradient (Sigma, #H8889). Aftercentrifugation (450×g, 30 minutes, room temperature), the plasma abovethe PBMC-containing interphase was discarded and PBMCs transferred in anew falcon tube subsequently filled with 50 ml of PBS. The mixture wascentrifuged (400×g, 10 minutes, room temperature), the supernatantdiscarded and the PBMC pellet washed twice with sterile PBS(centrifugation steps 350×g, 10 minutes). The resulting PBMC populationwas counted automatically (ViCell) and stored in RPMI1640 mediumcontaining 10% FCS and 1% L-alanyl-L-glutamine (Biochrom, K0302) at 37°C., 5% CO2 in cell incubator until further use (no longer than 24 h).For the killing assay, the antibody was added at the indicatedconcentrations (range of 0.005 pM-5 nM in triplicates). PBMCs were addedto target cells at final E:T ratio of 10:1. Target cell killing wasassessed after 24 h and 48 h of incubation at 37° C., 5% CO₂ byquantification of LDH released into cell supernatants byapoptotic/necrotic cells (LDH detection kit, Roche Applied Science, #11644 793 001). Maximal lysis of the target cells (=100%) was achieved byincubation of target cells with 1% Triton X-100. Minimal lysis (=0%)refers to target cells co-incubated with effector cells withoutbispecific construct.

The results show that the killing induced by 36F2 is strongly reduced incomparison to Mov19 TCB and 21A5 TCB (FIGS. 23A-B). The EC50 valuesrelated to killing assays, calculated using GraphPadPrism6 aresummarized in Table 27.

TABLE 27 EC50 values (pM) for T-cell mediated killing ofFolR1-expressing SKov-3 cells induced by 36F2 TCB, Mov19 TCB and 21A5TCB. EC50 [pM] Antibody 24 h 48 h 36F2 TCB 1406.07* 134.5 Mov19 TCB 0.750.05 21A5 TCB 2.83 0.10 *curve did not reach saturation, value ishypothetical

Example 33 T-Cell Killing Induced by 36F2 TCB and 16D5 TCB in DifferentMonovalent and Bivalent T-Cell Bispecific Formats

T-cell killing mediated by 36F2 TCB, 16D5 TCB, 16D5 TCB classical, 16D5TCB 1+1 and 16D5 TCB HT antibodies of Hela (high FolR1, about 2 millioncopies, Table 14, FIG. 27), Skov-3 (medium FolR1, about 70000-90000copies, Table 14, FIG. 27) and HT-29 (low FolR1, about 10000, Table 14,FIG. 27) human tumor cells was assessed. DP47 TCB antibody was includedas negative control. Human PBMCs were used as effectors and the killingwas detected at 24 h of incubation with the bispecific antibody.Briefly, target cells were harvested with Trypsin/EDTA, washed, andplated at density of 25 000 cells/well using flat-bottom 96-well plates.Cells were left to adhere overnight. Peripheral blood mononuclear cells(PBMCs) were prepared by Histopaque density centrifugation of enrichedlymphocyte preparations (buffy coats) obtained from healthy humandonors. Fresh blood was diluted with sterile PBS and layered overHistopaque gradient (Sigma, #H8889). After centrifugation (450×g, 30minutes, room temperature), the plasma above the PBMC-containinginterphase was discarded and PBMCs transferred in a new falcon tubesubsequently filled with 50 ml of PBS. The mixture was centrifuged(400×g, 10 minutes, room temperature), the supernatant discarded and thePBMC pellet washed twice with sterile PBS (centrifugation steps 350×g,10 minutes). The resulting PBMC population was counted automatically(ViCell) and stored in RPMI1640 medium containing 10% FCS and 1%L-alanyl-L-glutamine (Biochrom, K0302) at 37° C., 5% CO2 in cellincubator until further use (no longer than 24 h). For the killingassay, the antibody was added at the indicated concentrations (range of0.01 pM-100 nM in triplicates). PBMCs were added to target cells atfinal E:T ratio of 10:1. Target cell killing was assessed after 24 h ofincubation at 37° C., 5% CO₂ by quantification of LDH released into cellsupernatants by apoptotic/necrotic cells (LDH detection kit, RocheApplied Science, #11 644 793 001). Maximal lysis of the target cells(=100%) was achieved by incubation of target cells with 1% Triton X-100.Minimal lysis (=0%) refers to target cells co-incubated with effectorcells without bispecific construct.

The results show that target-specific killing of all three FolR1+ targetcell lines induced by 36F2 TCB is much weaker compared to the killinginduced by 16D5 TCB (FIGS. 24A-C, Table 29). Target-specific killinginduced by the monovalent 16D5 TCBs (16D5 HT and 16D5 1+1) is worsecompared to the bivalent 16D5 TCBs (16D5 TCB and 16D5 TCB classical).The EC50 values related to killing assays, calculated usingGraphPadPrism6, are summarized in Table 28. Importantly, this data showsthat using the 36F2 FolR1 binder in the bivalent 2+1 TCB format widensthe therapeutic window compared to the 16D5 FOLR1 TCB (FIG. 24A-C).Whereas the potency reduction between 16D5 and 36F2 FOLR1 TCB isapproximately 45-fold for Hela cells (high FOLR1 expression, see Table28: 16D5 TCB=0.8 versus 36F2 TCB 36.0) and approximately 297-fold forSkov3 cells (medium FOLR1 expression, see Table 28: 16D5 TCB=0.6 versus36F2 TCB 178.4), this reduction is almost 7000-fold for HT29 with lowFOLR1 expression (see Table 28: 16D5 TCB=5.7 versus 36F2 TCB 39573).Thus, the 36F2 FOLR1 TCB differentiates between high and low expressingcells which is of special importance to reduce toxicity as the cells ofsome normal, non-tumorous tissues express very low levels of FolR1(approximately less than 1000 copies per cell). Consistent with thisobservation, the results discussed in Example 35 below show that 36F2TCB does not induce T-cell killing of primary cells (FIGS. 26A-D)whereas for 16D5 TCB some killing can be observed on HRCEpiC and HRPEpiCcells after 48 h of incubation (FIGS. 26B and C). This importantcharacteristic of 36F2 TCB allows for dosing for the treatment ofFolR1-positive tumors so that it mediates potent killing of tumortissues with high or medium FOLR1 expression, but not of normal tissueswith low (partially polarized) expression. Notably, this characteristicappears to be mediated by the avidity of 36F2 TCB in the bivalent 2+1inverted format, as it was not observed when using the 1+1 monovalentformats carrying the same low affinity 36F2 binder.

Stated another way, 36F2 TCB in the bivalent 2+1 format comprises FolR1binding moieties of relatively low affinity but it possesses an avidityeffect which allows for differentiation between high and low FolR1expressing cells. Because tumor cells express FolR1 at high orintermediate levels, this TCB selectively binds to tumor cells and notnormal, non-cancerous cells that express FolR1 at low levels or not atall.

In addition to the above advantageous characteristics, the 36F2 TCB inthe bivalent 2+1 inverted format also has the advantage that it does notrequire chemical cross linking or other hybrid approach. This makes itsuitable for manufacture of a medicament to treat patients, for examplepatients having FolR1-positive cancerous tumors. The 36F2 TCB in thebivalent 2+1 inverted format can be produced using standard CHOprocesses with low aggregates. Further, the 36F2 TCB in the bivalent 2+1comprises human and humanized sequences making it superior to moleculesthat employ rat and murine polypeptides that are highly immunogenic whenadministered to humans. Furthermore, the 36F2 TCB in the bivalent 2+1format was engineered to abolish FcgR binding and, as such, does notcause FcgR crosslinking and infusion reactions, further enhancing itssafety when administered to patients.

As demonstrated by the results described above, its head-to-tailgeometry make the 36F2 TCB in the bivalent 2+1 inverted format a highlypotent molecule that induces absolute target cell killing. Its bivalencyenhance avidity and potency, but also allow for differentiation betweenhigh and low expressing cells. Its preference for high or medium targetexpressing cells due to its avidity affect reduce toxicity resultingfrom T cell mediated killing of normal cells that express FolR1 at lowlevels.

A further advantage of the 36F2 TCB in the bivalent 2+1 format and otherembodiments disclosed herein is that their clinical development does notrequire the use of surrogate molecules as they bind to human, cynomousand murine FolR1. As such, the molecules disclosed herein recognize adifferent epitope than antibodies to FolR1 previously described that donot recognize FolR1 from all three species.

TABLE 28 EC50 values (pM) for T-cell mediated killing ofFolR1-expressing tumor cells induced by 36F2 TCB and 16D5 TCB indifferent monovalent and bivalent T-cell bispecific formats after 24 hof incubation. Skov-3 Hela (FolR1 HT-29 Antibody (FolR1 high) medium)(FolR1 low) 16D5 TCB 0.8 0.6 5.7 16D5 TCB 4.6 2.0 13.0 classical 16D5TCB 11.6 12.3 15.1 HT 16D5 TCB 23.8 48.9 883.8* 1 + 1 36F2 TCB 36.0178.4 39573.0* *curve did not reach saturation, only hypothetical value

Table 29 shows a comparison of EC50 values of 16D5 TCB and 36F2 TCB onthe different cell lines tested. Out of the obtained EC50 values thedelta (EC50 of 16D5 TCB minus EC50 of 36F2 TCB) and the x-folddifference (EC50 of 16D5 TCB divided by the EC50 of 36F2 TCB) wascalculated.

TABLE 29 Comparison of EC50 values of 16D5 TCB and 36F2 TCB. Hela Skov-3HT-29 Antibody (FolR1 high) (FolR1 medium) (FolR1 low) 16D5 TCB 0.820.63 5.73 36F2 TCB 35.99 178.40 39573.00* Δ 35.17 177.77 39567.27 x-fold43.83 284.61 6906.58 *curve did not reach saturation, only hypotheticalvalue

The calculated EC50 values clearly show that the difference between 36F2TCB and 16D5 TCB gets larger the lower the FolR1 expression on thetarget cells is.

The same calculations as done for the comparison of the EC50 values of16D5 TCB and 36F2 TCB were done for 16D5 TCB and the two monovalent 16D5TCBs (16D5 TCB HT and 16D5 1+1). Tables 30 and 31 show the comparisonsof the EC50 values of 16D5 TCB vs 16D5 TCB HT (Table 30) and 16D5 TCB vs16D5 TCB 1+1 (Table 31) as well as the corresponding deltas (EC50 of16D5 TCB minus EC50 of 16D5 TCB HT/1+1) and the x-fold differences (EC50of 16D5 TCB divided by the EC50 of 16D5 TCB HT/1+1).

TABLE 30 Comparison of EC50 values of 16D5 TCB (2 + 1 inverted) and 16D5TCB HT. Hela Skov-3 HT-29 Antibody (FolR1 high) (FolR1 medium) (FolR1low) 16D5 TCB 0.82 0.63 5.73 16D5 TCB HT 11.61 12.27 15.11 Δ 10.79 11.659.38 x-fold 14.14 19.58 2.64

TABLE 31 Comparison of EC50 values of 16D5 TCB and 16D5 TCB 1 + 1. HelaSkov-3 HT-29 Antibody (FolR1 high) (FolR1 medium) (FolR1 low) 16D5 TCB0.82 0.63 5.73 16D5 TCB 1 + 1 23.84 48.86 883.78* Δ 23.02 48.24 878.05x-fold 29.03 77.95 154.24 *curve did not reach saturation, onlyhypothetical value

The comparison of the EC50 values of 16D5 TCB and 36F2 TCB (Table 29)shows that the difference in the EC50 values gets larger the lower theFolR1 expression on the target cells is. This effect cannot be seen inthe comparison of 16D5 TCB and the monovalent 16D5 TCBs (Table 29 andTable 30). For 16D5 TCB 1+1 (Table 31) there is also a slight increasein the difference between the EC50 of 16D5 TCB and 16D5 TCB 1+1 withdecreasing FolR1 expression but by far not as pronounced as can be seenin the comparison of 16D5 TCB vs 36F2 TCB.

Example 34 CD25 and CD69 Upregulation on CD8+ and CD4+Effector Cellsafter T Cell-Killing of FolR1-Expressing Tumor Cells Induced by 36F2 TCBand 16D5 TCB Antibody

Activation of CD8⁺ and CD4⁺ T cells after T-cell killing ofFolR1-expressing Hela, SKov-3 and HT-29 tumor cells mediated by 36F2 TCBand 16D5 TCB was assessed by FACS analysis using antibodies recognizingthe T cell activation markers CD25 (late activation marker) and CD69(early activation marker). DP47 TCB was included as non-binding control.The antibody and the killing assay conditions were essentially asdescribed above (Example 32) using the same antibody concentration range(0.01 pM-100 nM in triplicates), E:T ratio 10:1 and an incubation timeof 48 h.

After the incubation, PBMCs were transferred to a round-bottom 96-wellplate, centrifuged at 400×g for 4 min and washed twice with PBScontaining 0.1% BSA. Surface staining for CD8 (PE anti-human CD8, BD#555635), CD4 (Brilliant Violet 421™ anti-human CD4, Biolegend #300532),CD69 (FITC anti-human CD69, BD #555530) and CD25 (APC anti-human CD25 BD#555434) was performed according to the manufacturer's instructions.Cells were washed twice with 150 μl/well PBS containing 0.1% BSA. Aftercentrifugation, the samples were resuspended in 200 μl/well PBS 0.1% forthe FACS measurement. Samples were analyzed at BD FACS Canto II.

36F2 TCB induced a target-specific up-regulation of activation markers(CD25, CD69) on CD8+ and CD4+ T cells after killing of Hela (FIG. 25A)and SKov-3 (FIG. 25B) cells. In comparison to 16D5 TCB the up-regulationof CD25 and CD69 on CD8+ and CD4+ T cells induced by 36F2 is muchweaker.

On HT-29 (low FolR1) an up-regulation of activation markers can only beseen at the highest concentration of 36F2 TCB. In contrast, with 16D5TCB up-regulation of CD25 and CD69 can be seen already at much lowerantibody concentrations (FIG. 25C).

As seen as well in the tumor lysis experiment, the analysis ofactivation markers (CD25 and CD69) on T cells (CD4+ and CD8+) afterkilling clearly shows that the difference between 16D5 TCB and 36F2 TCBbecomes larger the lower the FolR1 expression level on the target cellsis.

Example 35 T-Cell Killing of Primary Cells Induced by 36F2 TCB and 16D5TCB

T-cell killing mediated by 36F2 TCB and 16D5 TCB was assessed on primarycells (Human Renal Cortical Epithelial Cells (HRCEpiC) (ScienCellResearch Laboratories; Cat No 4110) and Human Retinal Pigment EpithelialCells (HRPEpiC) (ScienCell Research Laboratories; Cat No 6540)). HT-29cells (low FolR1) were included as control cell line. DP47 TCB served asnon-binding control. Human PBMCs were used as effectors and the killingwas detected at 24 h and 48 h of incubation with the bispecificantibodies. Briefly, target cells were harvested with Trypsin/EDTA,washed, and plated at density of 25 000 cells/well using flat-bottom96-well plates. Cells were left to adhere overnight. Peripheral bloodmononuclear cells (PBMCs) were prepared by Histopaque densitycentrifugation of enriched lymphocyte preparations (buffy coats)obtained from healthy human donors. Fresh blood was diluted with sterilePBS and layered over Histopaque gradient (Sigma, #H8889). Aftercentrifugation (450×g, 30 minutes, room temperature), the plasma abovethe PBMC-containing interphase was discarded and PBMCs transferred in anew falcon tube subsequently filled with 50 ml of PBS. The mixture wascentrifuged (400×g, 10 minutes, room temperature), the supernatantdiscarded and the PBMC pellet washed twice with sterile PBS(centrifugation steps 350×g, 10 minutes). The resulting PBMC populationwas counted automatically (ViCell) and stored in RPMI1640 mediumcontaining 10% FCS and 1% L-alanyl-L-glutamine (Biochrom, K0302) at 37°C., 5% CO2 in cell incubator until further use (no longer than 24 h).For the killing assay, the antibody was added at the indicatedconcentrations (range of 0.01 pM-10 nM in triplicates). PBMCs were addedto target cells at final E:T ratio of 10:1. Target cell killing wasassessed after 24 h and 48 h of incubation at 37° C., 5% CO₂ byquantification of LDH released into cell supernatants byapoptotic/necrotic cells (LDH detection kit, Roche Applied Science, #11644 793 001). Maximal lysis of the target cells (=100%) was achieved byincubation of target cells with 1% Triton X-100. Minimal lysis (=0%)refers to target cells co-incubated with effector cells withoutbispecific construct.

The results show that 36F2 TCB does not induce T-cell killing of primarycells (FIG. 26A-D) whereas for 16D5 TCB some killing can be observed onHRCEpiC and HRPEpiC cells after 48 h of incubation (FIGS. 26B and D). Asdescribed above, a strong difference in T-cell killing between of HT-29cells was observed between 16D5 TCB and 36F2 TCB (FIG. 26E, F).

Example 36 Preparation of DP47 GS TCB (2+1 Crossfab-IgG P329G LALAInverted=“Untargeted TCB”)

The “untargeted TCB” was used as a control in the above experiments. Thebispecific antibody engages CD3e but does not bind to any other antigenand therefore cannot crosslink T cells to any target cells (andsubsequently cannot induce any killing). It was therefore used asnegative control in the assays to monitor any unspecific T cellactivation. This untargeted TCB was prepared as described inWO2014/131712. In brief, the variable region of heavy and light chainDNA sequences have been subcloned in frame with either the constantheavy chain or the constant light chain pre-inserted into the respectiverecipient mammalian expression vector. The antibody expression wasdriven by an MPSV promoter and carries a synthetic polyA signal sequenceat the 3′ end of the CDS. In addition each vector contains an EBV OriPsequence.

The molecule was produced by co-transfecting HEK293-EBNA cells with themammalian expression vectors using polyethylenimine. The cells weretransfected with the corresponding expression vectors in a 1:2:1:1 ratio(“vector heavy chain Fc(hole)”: “vector light chain”: “vector lightchain Crossfab”: “vector heavy chain Fc(knob)-FabCrossfab”).

For transfection HEK293 EBNA cells were cultivated in suspension serumfree in CD CHO culture medium. For the production in 500 ml shake flask400 million HEK293 EBNA cells were seeded 24 hours before transfection.For transfection cells were centrifuged for 5 min by 210×g, supernatantis replaced by pre-warmed 20 ml CD CHO medium. Expression vectors weremixed in 20 ml CD CHO medium to a final amount of 200 g DNA. Afteraddition of 540 μl PEI solution was vortexed for 15 s and subsequentlyincubated for 10 min at room temperature. Afterwards cells were mixedwith the DNA/PEI solution, transferred to a 500 ml shake flask andincubated for 3 hours by 37° C. in an incubator with a 5% CO2atmosphere. After incubation time 160 ml F17 medium was added and cellwere cultivated for 24 hours. One day after transfection 1 mM valporicacid and 7% Feed 1 was added. After 7 days cultivation supernatant wascollected for purification by centrifugation for 15 min at 210×g, thesolution was sterile filtered (0.22 μm filter) and sodium azide in afinal concentration of 0.01% w/v was added, and kept at 4° C.

The secreted protein was purified from cell culture supernatants byaffinity chromatography using ProteinA. Supernatant was loaded on aHiTrap ProteinA HP column (CV=5 mL, GE Healthcare) equilibrated with 40ml 20 mM sodium phosphate, 20 mM sodium citrate, 0.5 M sodium chloride,pH 7.5. Unbound protein was removed by washing with at least 10 columnvolume 20 mM sodium phosphate, 20 mM sodium citrate, 0.5 M sodiumchloride, pH 7.5. Target protein was eluted during a gradient over 20column volume from 20 mM sodium citrate, 0.5 M sodium chloride, pH 7.5to 20 mM sodium citrate, 0.5 M sodium chloride, pH 2.5. Protein solutionwas neutralized by adding 1/10 of 0.5 M sodium phosphate, pH 8. Targetprotein was concentrated and filtrated prior loading on a HiLoadSuperdex 200 column (GE Healthcare) equilibrated with 20 mM Histidine,140 mM sodium chloride solution of pH 6.0.

The protein concentration of purified protein samples was determined bymeasuring the optical density (OD) at 280 nm, using the molar extinctioncoefficient calculated on the basis of the amino acid sequence.

Purity and molecular weight of molecules were analyzed by CE-SDSanalyses in the presence and absence of a reducing agent. The CaliperLabChip GXII system (Caliper lifescience) was used according to themanufacturer's instruction. 2 ug sample is used for analyses.

The aggregate content of antibody samples was analyzed using a TSKgelG3000 SW XL analytical size-exclusion column (Tosoh) in 25 mM K2HPO4,125 mM NaCl, 200 mM L-Arginine Monohydrocloride, 0.02% (w/v) NaN3, pH6.7 running buffer at 25° C.

TABLE 32 Summary production and purification of DP47 GS TCB. Aggregateafter 1^(st) purification Mono- Titer Yield step HMW LMW mer Construct[mg/l] [mg/l] [%] [%] [%] [%] DP47 GS 103.7 8.04 8 2.3 6.9 91.8 TCB

TABLE 33 CE-SDS analyses of DP47 GS TCB. Peak kDa Corresponding ChainDP47 GS TCB non reduced 1 165.22 Molecule with 2 missing (A) lightchains 2 181.35 Molecule with 1 missing light chain 3 190.58 Correctmolecule without N-linked glycosylation 4 198.98 Correct molecule DP47GS TCB reduced (B) 1 27.86 Light chain DP47 GS 2 35.74 Light chainhuCH2527 3 63.57 Fc(hole) 4 93.02 Fc(knob)

Example 37 Binding of 16D5 TCB and 9D11 TCB and their Corresponding CD3Deamidation Variants N100A and S100aA to CD3-Expressing Jurkat Cells

The binding of 16D5 TCB and the corresponding CD3 deamidation variants16D5 TCB N100A and 16D5 TCB S100aA and 9D11 TCB and the demidationvariants 9D11 TCB N100A and 9D11 TCB S100aA to human CD3 was assessed ona CD3-expressing immortalized T lymphocyte line (Jurkat).

Briefly, cells were harvested, counted, checked for viability andresuspended at 2×10⁶ cells/ml in FACS buffer (100 μl PBS 0.1% BSA). 100μl of cell suspension (containing 0.2×10⁶ cells) was incubated inround-bottom 96-well plates for 30 min at 4° C. with differentconcentrations of the bispecific antibodies (686 pM-500 nM). After twowashing steps with cold PBS 0.1% BSA, samples were re-incubated forfurther 30 min at 4° C. with a PE-conjugated AffiniPure F(ab′)2 Fragmentgoat anti-human IgG Fcg Fragment Specific secondary antibody (JacksonImmuno Research Lab PE #109-116-170). After washing the samples twicewith cold PBS 0.1% BSA they were immediately analyzed by FACS using aFACS CantoII (Software FACS Diva). Binding curves were obtained usingGraphPadPrism6 (FIG. 28A-B).

The results show reduced binding of the deamidation variants N100A andS100aA to CD3 compared to the parental antibodies 16D5 TCB (FIG. 28A)and 9D11 TCB (FIG. 28B).

Example 38 T-Cell Killing of SKov-3 and HT-29 Cells Induced by 16D5 TCBand 9D11 TCB and their CD3 Deamidation Variants N100A and S100aA

T-cell killing mediated by 16D5 TCB and the corresponding CD3deamidation variants 16D5 TCB N100A and 16D5 TCB S100aA and 9D11 TCB andthe demidation variants 9D11 TCB N100A and 9D11 TCB S100aA was assessedon SKov-3 (medium FolR1) and HT-29 (low FolR1) cells. Human PBMCs wereused as effectors and the killing was detected at 24 h of incubationwith the bispecific antibodies. Briefly, target cells were harvestedwith Trypsin/EDTA, washed, and plated at a density of 25 000 cells/wellusing flat-bottom 96-well plates. Cells were left to adhere overnight.Peripheral blood mononuclear cells (PBMCs) were prepared by Histopaquedensity centrifugation of enriched lymphocyte preparations (buffy coats)obtained from healthy human donors. Fresh blood was diluted with sterilePBS and layered over Histopaque gradient (Sigma, #H8889). Aftercentrifugation (450×g, 30 minutes, room temperature), the plasma abovethe PBMC-containing interphase was discarded and PBMCs transferred in anew falcon tube subsequently filled with 50 ml of PBS. The mixture wascentrifuged (400×g, 10 minutes, room temperature), the supernatantdiscarded and the PBMC pellet washed twice with sterile PBS(centrifugation steps 350×g, 10 minutes). The resulting PBMC populationwas counted automatically (ViCell) and stored in RPMI1640 mediumcontaining 10% FCS and 1% L-alanyl-L-glutamine (Biochrom, K0302) at 37°C., 5% CO2 in cell incubator until further use (no longer than 24 h).For the killing assay, the antibody was added at the indicatedconcentrations (range of 0.01 pM-10 nM in triplicates). PBMCs were addedto target cells at final E:T ratio of 10:1. Target cell killing wasassessed after 24 h of incubation at 37° C., 5% CO₂ by quantification ofLDH released into cell supernatants by apoptotic/necrotic cells (LDHdetection kit, Roche Applied Science, #11 644 793 001). Maximal lysis ofthe target cells (=100%) was achieved by incubation of target cells with1% Triton X-100. Minimal lysis (=0%) refers to target cells co-incubatedwith effector cells without bispecific construct.

The results show that on SKov-3 cells the killing induced by the CD3deamidation variants 16D5 TCB N100A and 16D5 S100aA is comparable to theone induced by 16D5 TCB (FIG. 29A). The same is true for 9D11 TCB andits variants 9D11 TCB N100A and 9D11 TCB S100aA (FIG. 29B). On FolR1 lowexpressing HT-29 cells the S100aA variant shows an impaired killingefficiency which is the case for 16D5 TCB (FIG. 30A) as well as for 9D11TCB (FIG. 30B). The EC50 values related to killing assays, calculatedusing GraphPadPrism6 are given in Table 35.

TABLE 35 EC50 values (pM) for T-cell mediated killing ofFolR1-expressing SKov-3 and HT-29 cells induced by 16D5 TCB and 9D11 TCBand their deamidation variants N100A and A100aA. EC50 [pM] AntibodySKov-3 HT-29 16D5 TCB 1.283 56.67 16D5 TCB 1.886 91.95 N100A 16D5 TCB1.939 165.6 S100aA 9D11 TCB 1.283 2.827 9D11 TCB 1.886 37.72 N100A 9D11TCB 1.939 n.d.* S100aA *not determined

Example 39 Biochemical Characterization by Surface Plasmon Resonance asTCBs of Two CD3 Binder Variants (N100A and S100aA) to Remove aDeamidation Site

Binding of two 16D5 TCBs with CD3 binder variants (N100A or S100aA) tohuman recombinant CD3 (CD3epsilon-CD3delta heterodimer as Fc fusion) wasassessed by surface plasmon resonance (SPR). All SPR experiments wereperformed on a Biacore T200 at 25° C. with HBS-EP as running buffer(0.01 M HEPES pH 7.4, 0.15 M NaCl, 3 mM EDTA, 0.005% Surfactant P20,Biacore, Freiburg/Germany).

Affinity to CD3ed-Fc

The affinity of the interaction between the anti-FolR1 T cellbispecifics and the recombinant CD3 epsilon-delta heterodimer wasdetermined as described below (Table 36).

For affinity measurement, direct coupling of around 6000 resonance units(RU) of the anti-human Fab specific antibody (Fab capture kit, GEHealthcare) was performed on a CM5 chip at pH 5.0 using the standardamine coupling kit (GE Healthcare). Anti-FolR1 T cell bispecifics werecaptured at 200 nM with a flow rate of 20 μl/min for 60 sec, thereference flow cell was left without capture. Dilution series (4.1 to3000 nM) of human and cyno Folate Receptor 1 Fc fusion were passed onall flow cells at 30 μl/min for 240 sec to record the association phase.The dissociation phase was monitored for 240 s and triggered byswitching from the sample solution to HBS-EP. The chip surface wasregenerated after every cycle using a double injection of 60 sec 10 mMGlycine-HCl pH 1.5. Bulk refractive index differences were corrected forby subtracting the response obtained on the reference flow cell 1. Theaffinity constants for the interactions were derived from the rateconstants by fitting to a 1:1 Langmuir binding using the Bia Evaluationsoftware (GE Healthcare).

TABLE 36 Monovalent binding (affinity) of two 16D5 CD3 deamidationvariants as TCBs on human CD3ed-Fc. Ligand Analyte ka (1/Ms) kd (1/s) KD16D5 TCB N100A huCD3 1.23E+04 4.67E−03 380 nM 16D5 TCB S100aA huCD31.21E+04 5.49E−03 460 nM 16D5 TCB huCD3 2.03E+04 4.41E−03 220 nM

The two CD3 deamidation variants have a slightly reduced affinitycompared to the wild-type CD3 binder (CH2527), but the difference is notgrave.

Example 40 Production and Purification of Two Variants of the 16D5T-Cell Bispecific with Mutations to Remove the Deamidation Site in theCD3 Binder: 16D5 TCB N100A, 16D5 TCB S100aA

Transient Transfection and Production

The two deamidation variants 16D5 TCBs were transiently produced inHEK293 EBNA cells using a PEI mediated transfection procedure for therequired vectors as described below. HEK293 EBNA cells are cultivated insuspension serum free in CD CHO culture medium. For the production in500 ml shake flask 400 million HEK293 EBNA cells are seeded 24 hoursbefore transfection (for alternative scales all amounts were adjustedaccordingly). For transfection cells are centrifuged for 5 min by 210×g,supernatant is replaced by pre-warmed 20 ml CD CHO medium. Expressionvectors are mixed in 20 ml CD CHO medium to a final amount of 200 μgDNA. After addition of 540 μl PEI solution is vortexed for 15 s andsubsequently incubated for 10 min at room temperature. Afterwards cellsare mixed with the DNA/PEI solution, transferred to a 500 ml shake flaskand incubated for 3 hours by 37° C. in an incubator with a 5% CO2atmosphere. After incubation time 160 ml F17 medium is added and cellare cultivated for 24 hours. One day after transfection 1 mM valporicacid and 7% Feed 1 is added. After 7 days cultivation supernatant iscollected for purification by centrifugation for 15 min at 210×g, thesolution is sterile filtered (0.22 μm filter) and sodium azide in afinal concentration of 0.01% w/v is added, and kept at 4° C. Afterproduction the supernatant was harvested, filtered through 0.22 μmsterile filters and stored at 4° C. until purification.

Purification

The two deamidation variants 16D5 TCBs were purified in two steps usingstandard procedures, such as protein A affinity purification (ÄktaExplorer) and size exclusion chromatography. The supernatant obtainedfrom transient production was adjusted to pH 8.0 (using 2 M TRIS pH 8.0)and applied to Mab Select SuRe (GE Healthcare, column volume (cv)=2 ml)equilibrated with 8 column volumes (cv) buffer A (20 mM sodium phosphatepH 7.5, 20 mM sodium citrate). After washing with 10 cv of buffer A, theprotein was eluted using a pH gradient to buffer B (20 mM sodium citratepH 3.0, 100 mM NaCl, 100 mM glycine) over 20 cv. Fractions containingthe protein of interest were pooled and the pH of the solution wasgently adjusted to pH 6.0 (using 0.5 M Na₂HPO₄ pH 8.0). Samples wereconcentrated to 1 ml using ultra-concentrators (Amicon Ultra-15, 30.000MWCO, Millipore) and subsequently applied to a HiLoad™ 16/60 Superdex™200 preparative grade (GE Healthcare) equilibrated with 20 mM Histidine,pH 6.0, 140 mM NaCl. The aggregate content of eluted fractions wasanalyzed by analytical size exclusion chromatography. Therefore, 30 μlof each fraction was applied to a TSKgel G3000 SW XL analyticalsize-exclusion column (Tosoh) equilibrated in 25 mM K₂HPO₄, 125 mM NaCl,200 mM L-arginine monohydrochloride, 0.02% (w/v) NaN₃, pH 6.7 runningbuffer at 25° C. Fractions containing less than 2% oligomers werepooled. The protein concentration was determined by measuring theoptical density (OD) at 280 nm, using the molar extinction coefficientcalculated on the basis of the amino acid sequence. Purity and molecularweight of the constructs were analyzed by SDS capillary electrophoresis(CE-SDS) in the presence and absence of a reducing agent following themanufacturer instructions (instrument Caliper LabChipGX, Perkin Elmer).Purified proteins were frozen in liquid N₂ and stored at −80° C.

TABLE 36 Yield, monomer content and purity by CE-SDS of the two 16D5deamidation variants in the T cell bispecific format. Mutation in theMonomer Purity by CE- Name CD3 binder Yield [mg/L] [%] SDS [%] 16D5 TCBN100A 9 100 89 16D5 TCB S100aA 23 100 83 16D5 TCB Wild-type 9 100 93

Both TCBs were produced in good quality, similar to the construct withthe wild-type CD3 binder.

Example 41 Production and Purification of Two 16D5 Binder Variants(D52dE and D52dQ) as IgGs to Remove a Hotspot in the CDR

Transient Transfection and Production

The two IgGs were transiently produced in HEK293 EBNA cells using a PEImediated transfection procedure for the required vectors as describedbelow. HEK293 EBNA cells are cultivated in suspension serum free in CDCHO culture medium. For the production in 500 ml shake flask 400 millionHEK293 EBNA cells are seeded 24 hours before transfection (foralternative scales all amounts were adjusted accordingly). Fortransfection cells are centrifuged for 5 min by 210×g, supernatant isreplaced by pre-warmed 20 ml CD CHO medium. Expression vectors are mixedin 20 ml CD CHO medium to a final amount of 200 μg DNA. After additionof 540 μl PEI solution is vortexed for 15 s and subsequently incubatedfor 10 min at room temperature. Afterwards cells are mixed with theDNA/PEI solution, transferred to a 500 ml shake flask and incubated for3 hours by 37° C. in an incubator with a 5% CO2 atmosphere. Afterincubation time 160 ml F17 medium is added and cell are cultivated for24 hours. One day after transfection 1 mM valporic acid and 7% Feed 1 isadded. After 7 days cultivation supernatant is collected forpurification by centrifugation for 15 min at 210×g, the solution issterile filtered (0.22 μm filter) and sodium azide in a finalconcentration of 0.01% w/v is added, and kept at 4° C. After productionthe supernatants were harvested and the antibody containing supernatantswere filtered through 0.22 μm sterile filters and stored at 4° C. untilpurification.

Antibody Purification

The two IgGs were purified in two steps using standard procedures, suchas protein A affinity purification (Äkta Explorer) and size exclusionchromatography. The supernatant obtained from transient production wasadjusted to pH 8.0 (using 2 M TRIS pH 8.0) and applied to POROSMabCapture A (Applied Biosystems, column volume (cv)=1 ml) equilibratedwith 8 column volumes (cv) buffer A (20 mM sodium phosphate, 20 mMsodium citrate, pH 7.5). After washing with 10 cv of buffer A, theprotein was eluted using a pH step to buffer B (20 mM sodium citrate pH3.0, 100 mM NaCl, 100 mM glycine) over 5 cv. The 5 ml containing theprotein of interest are stored in a loop on the Äkta Explorer andsubsequently applied to a HiLoad 16/60 Superdex™ 200 (GE Healthcare)equilibrated with 20 mM Histidine, pH 6.0, 140 mM NaCl (no TWEEn wasused). Fractions containing the IgGs were pooled and concentrated usingultra concentrators (Amicon Ultra-15, 30.000 MWCO, Millipore). Theaggregate content of the final pool was analyzed by analytical sizeexclusion chromatography. Therefore, 30 μl were applied to a TSKgelG3000 SW XL analytical size-exclusion column (Tosoh) equilibrated in 25mM K₂HPO₄, 125 mM NaCl, 200 mM L-arginine monohydrochloride, 0.02% (w/v)NaN₃, pH 6.7 running buffer at 25° C. The protein concentration wasdetermined by measuring the optical density (OD) at 280 nm, using themolar extinction coefficient calculated on the basis of the amino acidsequence. Purity and molecular weight of the constructs were analyzed bySDS capillary electrophoresis (CE-SDS) in the presence and absence of areducing agent following the manufacturer instructions (instrumentCaliper LabChipGX, Perkin Elmer). Purified proteins were stored at 4° C.

TABLE 37 Yield, monomer content and purity by CE-SDS of two 16D5 IgGhotspot variants. Mutation Monomer Purity by CE- Clone HC/LC Yield[mg/L} [%] SDS [%] 16D5 D52dE 9 100 96 16D5 D52dQ 20 100 96

Both IgGs produced well and in good quality.

Example 42 Biochemical Characterization by Surface Plasmon Resonance ofTwo 16D5 Binder Variants (D52dE and D52dQ) as IgGs to Remove a Hotspotin the CDR

Binding of two 16D5 binder variants (D52dE and D52dQ) as IgGs to humanand cyno recombinant folate receptor 1 (both as Fc fusions) was assessedby surface plasmon resonance (SPR). All SPR experiments were performedon a Biacore T200 at 25° C. with HBS-EP as running buffer (0.01 M HEPESpH 7.4, 0.15 M NaCl, 3 mM EDTA, 0.005% Surfactant P20, Biacore,Freiburg/Germany).

1. Avidity to Folate Receptor 1

The avidity of the interaction between the anti-FolR1 IgGs or T cellbispecifics and the recombinant folate receptors was determined asdescribed below (Table 38).

Recombinant biotinylated monomeric Fc fusions of human, cynomolgus andmurine Folate Receptor 1 (FolR1-Fc) were directly coupled on a SA chipusing the standard coupling instruction (Biacore, Freiburg/Germany). Theimmobilization level was about 160. The anti-FolR1 IgGs or T cellbispecifics were passed at a concentration range from 3.7 to 900 nM witha flow of 30 μL/minutes through the flow cells over 180 seconds. Thedissociation was monitored for 600 seconds. Bulk refractive indexdifferences were corrected for by subtracting the response obtained onreference flow cell immobilized with recombinant biotinylated murine IL2receptor Fc fusion. The binding curves resulting from the bivalentbinding of the IgG or T cell bispecifics were approximated to a 1:1Langmuir binding (even though it is a 1:2 binding) and fitted with thatmodel to get an apparent KD representing the avidity of the bivalentbinding. The apparent avidity constants for the interactions werederived from the rate constants of the fitting using the Bia Evaluationsoftware (GE Healthcare).

TABLE 38 Bivalent binding (avidity with apparent KD) of two 16D5 hotspot variants as IgGs on human, murine and cyno FolR1 (no binding onmuFolR1 as expected). Analyte Ligand ka (1/Ms) kd (1/s) Apparent KD 16D5D52dE IgG huFolR1 1.62E+05 5.45E−04 3.4 nM cyFolR1 2.98E+06 7.47E−03 2.5nM 16D5 D52dQ IgG huFolR1 8.40E+04 7.75E−04 9.2 nM cyFolR1 4.12E+052.04E−03   5 nM 16D5 TCB huFolR1 2.25E+05 5.00E−04 2.2 nM cyFolR12.71E+05 6.63E−04 2.5 nM

2. Affinity to Folate Receptor 1

The affinity of the interaction between the anti-FolR1 IgGs or the Tcell bispecifics and the recombinant folate receptors was determined asdescribed below (Table 39).

For affinity measurement, direct coupling of around 10000 resonanceunits (RU) of the anti-human Fab specific antibody (Fab capture kit, GEHealthcare) was performed on a CM5 chip at pH 5.0 using the standardamine coupling kit (GE Healthcare). Anti-FolR1 IgGs or T cellbispecifics were captured at 20 nM with a flow rate of 10 μl/min for 40sec, the reference flow cell was left without capture. Dilution series(12.35 to 3000 nM) of human and cyno Folate Receptor 1 Fc fusion werepassed on all flow cells at 30 μl/min for 240 sec to record theassociation phase. The dissociation phase was monitored for 300 s andtriggered by switching from the sample solution to HBS-EP. The chipsurface was regenerated after every cycle using a double injection of 60sec 10 mM Glycine-HCl pH 1.5. Bulk refractive index differences werecorrected for by subtracting the response obtained on the reference flowcell 1. The affinity constants for the interactions were derived fromthe rate constants by fitting to a 1:1 Langmuir binding using the BiaEvaluation software (GE Healthcare).

TABLE 39 Monovalent binding (affinity) of two 16D5 hot spot variants asIgGs on human and cyno FolR1. Ligand Analyte ka (1/Ms) kd (1/s) KD 16D5D52dE IgG huFolR1 2.40E+04 2.27E−03  95 nM cyFolR1 2.25E+04 1.20E−02 530nM 16D5 D52dQ IgG huFolR1 6.97E+03 1.62E−03 230 nM cyFolR1 8.20E+033.32E−03 410 nM 16D5 TCB huFolR1 2.05E+04 7.05E−04  35 nM cyFolR11.72E+04 1.62E−03  90 nM

The two 16D5 hot spot variants have similar avidity (bivalent binding)than the wild-type 16D5 binder. The avidity is slightly decreased forthe D52dQ variant and this difference is even more visible in affinity(monovalent binding).

Example 43 Production and Purification as IgGs of Twelve Variants of the16D5 Binder with Mutations in the Heavy and Light Chain to ReduceAffinity to FolR1

Transient Transfection and Production

The twelve IgGs were transiently produced in HEK293 EBNA cells using aPEI mediated transfection procedure for the required vectors asdescribed below. HEK293 EBNA cells are cultivated in suspension serumfree in CD CHO culture medium. For the production in 500 ml shake flask400 million HEK293 EBNA cells are seeded 24 hours before transfection(for alternative scales all amounts were adjusted accordingly). Fortransfection cells are centrifuged for 5 min by 210×g, supernatant isreplaced by pre-warmed 20 ml CD CHO medium. Expression vectors are mixedin 20 ml CD CHO medium to a final amount of 200 μg DNA. After additionof 540 μl PEI solution is vortexed for 15 s and subsequently incubatedfor 10 min at room temperature. Afterwards cells are mixed with theDNA/PEI solution, transferred to a 500 ml shake flask and incubated for3 hours by 37° C. in an incubator with a 5% CO2 atmosphere. Afterincubation time 160 ml F17 medium is added and cell are cultivated for24 hours. One day after transfection 1 mM valporic acid and 7% Feed 1 isadded. After 7 days cultivation supernatant is collected forpurification by centrifugation for 15 min at 210×g, the solution issterile filtered (0.22 μm filter) and sodium azide in a finalconcentration of 0.01% w/v is added, and kept at 4° C. After productionthe supernatants were harvested and the antibody containing supernatantswere filtered through 0.22 μm sterile filters and stored at 4° C. untilpurification.

Antibody Purification

All molecules were purified in two steps using standard procedures, suchas protein A affinity purification (Äkta Explorer) and size exclusionchromatography. The supernatant obtained from transient production wasadjusted to pH 8.0 (using 2 M TRIS pH 8.0) and applied to POROSMabCapture A (Applied Biosystems, column volume (cv)=1 ml) equilibratedwith 8 column volumes (cv) buffer A (20 mM sodium phosphate, 20 mMsodium citrate, pH 7.5). After washing with 10 cv of buffer A, theprotein was eluted using a pH step to buffer B (20 mM sodium citrate pH3.0, 100 mM NaCl, 100 mM glycine) over 5 cv. The 5 ml containing theprotein of interest are stored in a loop on the Äkta Explorer andsubsequently applied to a HiLoad 16/60 Superdex™ 200 (GE Healthcare)equilibrated with 20 mM Histidine, pH 6.0, 140 mM NaCl, 0.01% Tween-20.Fractions containing the IgGs were pooled and concentrated using ultraconcentrators (Amicon Ultra-15, 30.000 MWCO, Millipore). The aggregatecontent of the final pool was analyzed by analytical size exclusionchromatography. Therefore, 30 μl were applied to a TSKgel G3000 SW XLanalytical size-exclusion column (Tosoh) equilibrated in 25 mM K₂HPO₄,125 mM NaCl, 200 mM L-arginine monohydrochloride, 0.02% (w/v) NaN₃, pH6.7 running buffer at 25° C. The protein concentration was determined bymeasuring the optical density (OD) at 280 nm, using the molar extinctioncoefficient calculated on the basis of the amino acid sequence. Purityand molecular weight of the constructs were analyzed by SDS capillaryelectrophoresis (CE-SDS) in the presence and absence of a reducing agentfollowing the manufacturer instructions (instrument Caliper LabChipGX,Perkin Elmer). Purified proteins were stored at 4° C.

TABLE 40 Yield, monomer content and purity by CE-SDS of twelve 16D5 IgGvariants Mutation Monomer Purity by CE- Clone HC/LC Yield [mg/L} [%] SDS[%] 16D5 W98Y/wt 32 100 100 16D5 W98Y/K53A 24 100 100 16D5 S35H/wt 21100 100 16D5 S35H/K53A 18 100 100 16D5 S35H/S93A 18 100 100 16D5 W96Y/wt40 100 100 16D5 W96Y/K53A 21 100 100 16D5 W96Y/S93A 25 98 100 16D5R50S/K53A 10 98 100 16D5 R50S/S93A 7 100 100 16D5 G49S/K53A 42 100 10016D5 G49S/S93A 45 100 100

All twelve IgGs produced well and in good quality.

Example 44 Biochemical Characterization of 16D5 Heavy and Light ChainCombination Variants as IgG by Surface Plasmon Resonance

Binding of FolR1 16D5 heavy and light chain combination variants bindersas IgG to different recombinant folate receptors (human, murine andcynomolgus FolR1; all as Fc fusions) was assessed by surface plasmonresonance (SPR). All SPR experiments were performed on a Biacore T200 at25° C. with HBS-EP as running buffer (0.01 M HEPES pH 7.4, 0.15 M NaCl,3 mM EDTA, 0.005% Surfactant P20, Biacore, Freiburg/Germany).

Avidity to Folate Receptor 1

The avidity of the interaction between the anti-FolR1 IgGs or T cellbispecifics and the recombinant folate receptors was determined asdescribed below (Table 41).

Recombinant biotinylated monomeric Fc fusions of human, cynomolgus andmurine Folate Receptor 1 (FolR1-Fc) were directly coupled on a SA chipusing the standard coupling instruction (Biacore, Freiburg/Germany). Theimmobilization level was about 300. The anti-FolR1 IgGs or T cellbispecifics were passed at a concentration range from 11.1 to 900 nMwith a flow of 30 μL/minutes through the flow cells over 180 seconds.The dissociation was monitored for 240 or 600 seconds. Bulk refractiveindex differences were corrected for by subtracting the responseobtained on reference flow cell immobilized with recombinantbiotinylated murine IL2 receptor Fc fusion. The binding curves resultingfrom the bivalent binding of the IgG or T cell bispecifics wereapproximated to a 1:1 Langmuir binding (even though it is a 1:2 binding)and fitted with that model to get an apparent KD representing theavidity of the bivalent binding. The apparent avidity constants for theinteractions were derived from the rate constants of the fitting usingthe Bia Evaluation software (GE Healthcare). For low affinity kineticswith association and dissociation phases too fast to be fitted by the1:1 Langmuir binding model, the steady state analysis model was appliedusing the Bia Evaluation software (GE Healthcare). The steady stateanalysis gives the KD of the binding reaction at equilibrium.

TABLE 41 Bivalent binding (avidity with apparent KD) of twelve 16D5variants binders as IgGs on human, murine and cyno FolR1. Analyte HCvariant/LC Apparent variant Ligand ka (1/Ms) kd (1/s) KD W98Y/K53AhuFolR1 Weak binding cyFolR1 Weak binding S35H/K53A huFolR1 2.10E+042.91E−02 1400 nM  cyFolR1 3.47E+04 4.04E−02 1100 nM  W96Y/K53A huFolR1580 nM  (steady state) cyFolR1 660 nM  (steady state) W98Y/wt huFolR11.36E+05 3.28E−02 240 nM  cyFolR1 1.71E+05 3.61E−02 200 nM  S35H/S93AhuFolR1 2.43E+05 2.20E−02 90 nM cyFolR1 6.12E+05 6.77E−02 110 nM G49S/K53A huFolR1 1.90E+05 1.15E−02 60 nM cyFolR1 3.93E+05 3.28E−02 80nM R50S/K53A huFolR1 3.28E+05 1.97E−02 60 nM cyFolR1 5.50E+05 4.55E−0280 nM S35H/wt huFolR1 1.32E+05 5.68E−03 40 nM cyFolR1 2.23E+05 1.24E−0255 nM R50S/S93A huFolR1 1.25E+05 3.23E−03 30 nM cyFolR1 4.39E+057.80E−03 20 nM W96Y/S93A huFolR1 6.55E+05 1.89E−02 30 nM cyFolR16.25E+05 1.74E−02 30 nM G49S/S93A huFolR1 1.52E+05 3.06E−03 20 nMcyFolR1 3.58E+05 6.22E−03 20 nM W96Y/wt huFolR1 1.29E+05 2.13E−03 20 nMcyFolR1 1.73E+05 2.11E−03 10 nM 36F2 TCB huFolR1 2.44E+06 1.37E−02  6 nMcyFolR1 4.12E+06 2.15E−02  5 nM muFolR1 4.86E+05 1.20E−03 2.5 nM  16D5TCB huFolR1 1.41E+05 4.25E−04  3 nM cyFolR1 1.78E+05 6.39E−04 3.5 nM 

Affinity to Folate Receptor 1

The affinity of the interaction between the anti-FolR1 IgGs or the Tcell bispecifics and the recombinant folate receptors was determined asdescribed below (Table 42).

For affinity measurement, direct coupling of around 10000 resonanceunits (RU) of the anti-human Fab specific antibody (Fab capture kit, GEHealthcare) was performed on a CM5 chip at pH 5.0 using the standardamine coupling kit (GE Healthcare). Anti-FolR1 IgGs or T cellbispecifics were captured at 200 nM with a flow rate of 10 ul/min for 40sec, the reference flow cell was left without capture. Dilution series(12.35 to 3000 nM) of human Folate Receptor 1 Fc fusion were passed onall flow cells at 30 μl/min for 240 sec to record the association phase.The dissociation phase was monitored for 300 s and triggered byswitching from the sample solution to HBS-EP. The chip surface wasregenerated after every cycle using a double injection of 60 sec 10 mMGlycine-HCl pH 1.5. Bulk refractive index differences were corrected forby subtracting the response obtained on the reference flow cell 1. Theaffinity constants for the interactions were derived from the rateconstants by fitting to a 1:1 Langmuir binding using the Bia Evaluationsoftware (GE Healthcare).

TABLE 42 Monovalent binding (affinity) of twelve 16D5 variants FolR1binders as IgGs on human, cyno and murine FolR1. Ligand HC variant/LCvariant Analyte ka (1/Ms) kd (1/s) KD W98Y/K53A huFolR1 No bindingS35H/K53A huFolR1 Weak binding W96Y/K53A huFolR1 Weak binding W98Y/wthuFolR1 5400 nM (steady state) G49S/K53A huFolR1 9.19E+03 1.74E−02 1900nM R50S/K53A huFolR1 1.35E+04 2.45E−02 1800 nM 36F2 TCB huFolR1 5.00E+048.57E−02 1700 nM S35H/S93A huFolR1 8.43E+03 1.12E−02 1300 nM S35H/wthuFolR1 8.96E+03 1.13E−02 1200 nM R50S/S93A huFolR1 1.57E+04 1.23E−02 780 nM G49S/S93A huFolR1 1.05E+04 7.99E−03  760 nM W96Y/wt huFolR19.95E+03 5.44E−03  550 nM W96Y/S93A huFolR1 4.05E+04 1.72E−02  420 nM16D5 TCB huFolR1 1.18E+04 7.22E−04  60 nM

Twelve “affinity reduced” variants of the 16D5 FolR1 binder wereanalyzed by surface plasmon resonance in comparison to the 16D5wild-type binder and the 36F2 binder. The goal was to find a 16D5variant with an affinity and an avidity comparable to 36F2. Whenmeasuring monovalent binding (affinity) there were variants with ahigher and variants with a lower affinity than 36F2. However in thebivalent binding (avidity) all the variants have a higher apparent KDvalue than 36F2. This is mainly due to the fast association rate (ka) of36F2 that results in a small apparent KD for 36F2. The big avidityeffect when 36F2 binds bivalently seems to be unique to this binder. Asnoted above, 36F2 was the only human, murine and cyno crossreactivebinder that could be identified.

Example 45 Binding of 16D5 HC/LC Variants to Human FolR1 Expressed onHela Cells

The binding of 36F2 TCB, 16D5 TCB and various HC/LC variants of 16D5 tohuman FolR1 was assessed on Hela cells. Briefly, cells were harvested,counted, checked for viability and resuspended at 2×10⁶ cells/ml in FACSbuffer (100 μl PBS 0.1% BSA). 100 μl of cell suspension (containing0.2×10⁶ cells) was incubated in round-bottom 96-well plates for 30 minat 4° C. with different concentrations of the bispecific antibodies (229pM-500 nM). After two washing steps with cold PBS 0.1% BSA, samples werere-incubated for further 30 min at 4° C. with a PE-conjugated AffiniPureF(ab′)2 Fragment goat anti-human IgG Fcg Fragment Specific secondaryantibody (Jackson Immuno Research Lab PE #109-116-170). After washingthe samples twice with cold PBS 0.1% BSA they were fixed with 1% PFAovernight. Afterwards samples were centrifuged, resuspended in PBS 0.1%BSA and analyzed by FACS using a FACS CantoII (Software FACS Diva).Binding curves were obtained using GraphPadPrism6 (FIG. 32A-E). The 36F2TCB bound FolR2, was not well tolerated in mice, and did not demonstratethe desired efficacy.

Example 46 Production and Purification of Four Variants of the 16D5T-Cell Bispecific with Mutations to Reduce the Affinity to Human andCynomolgus FolR1: 16D5 TCB G49S/S93A, G49S/K53A, W96Y, W96Y/D52E

Transient Transfection and Production

Four additional variants of 16D5 TCBs having reduced affinity to FolR1were transiently produced in HEK293 EBNA cells using a PEI mediatedtransfection procedure for the required vectors as described below. Fortransfection HEK293 EBNA cells are cultivated in suspension serum freein Excell culture medium containing 6 mM L-Glutamine and 250 mg/l G418culture medium. For the production in 600 ml tubespin flask (max.working volume 400 mL) 600 million HEK293 EBNA cells are seeded 24 hoursbefore transfection. For transfection cells are centrifuged for 5 min by210×g, supernatant is replaced by pre-warmed 20 ml CD CHO medium.Expression vectors are mixed in 20 ml CD CHO medium to a final amount of400 μg DNA. After addition of 1080 μl PEI solution (2.7 μg/ml) isvortexed for 15 s and subsequently incubated for 10 min at roomtemperature. Afterwards cells are mixed with the DNA/PEI solution,transferred to a 600 ml tubespin flask and incubated for 3 hours by 37°C. in an incubator with a 5% CO2 atmosphere. After incubation time 360ml Excell+6 mM L-Glutamine+5 g/L Pepsoy+1.0 mM VPA medium is added andcells are cultivated for 24 hours. One day after transfection 7% Feed 7is added. After 7 days cultivation supernatant is collected forpurification by centrifugation for 20-30 min at 3600×g (Sigma 8Kcentrifuge), the solution is sterile filtered (0.22 mm filter) andsodium azide in a final concentration of 0.01% w/v is added, and kept at4° C.

Purification

The reduced affinity variants 16D5 TCBs were purified in two steps usingstandard procedures, such as protein A affinity purification (ÄktaExplorer) and size exclusion chromatography. The supernatant obtainedfrom transient production was adjusted to pH 8.0 (using 2 M TRIS pH 8.0)and applied to HiTrap Protein A (GE Healthcare, column volume (cv)=5 ml)equilibrated with 8 column volumes (cv) buffer A (20 mM sodium phosphatepH 7.5, 20 mM sodium citrate). After washing with 10 cv of buffer A, theprotein was eluted using a pH gradient to buffer B (20 mM sodium citratepH 3.0, 100 mM NaCl, 100 mM glycine) over 20 cv. Fractions containingthe protein of interest were pooled and the pH of the solution wasgently adjusted to pH 6.0 (using 0.5 M Na₂HPO₄ pH 8.0). Samples wereconcentrated to 1 ml using ultra-concentrators (Amicon Ultra-15, 30.000MWCO, Millipore) and subsequently applied to a HiLoad™ 16/60 Superdex™200 preparative grade (GE Healthcare) equilibrated with 20 mM Histidine,pH 6.0, 140 mM NaCl, 0.01% Tween 20. The aggregate content of elutedfractions was analyzed by analytical size exclusion chromatography.Therefore, 30 μl of each fraction was applied to a TSKgel G3000 SW XLanalytical size-exclusion column (Tosoh) equilibrated in 25 mM K₂HPO₄,125 mM NaCl, 200 mM L-arginine monohydrochloride, 0.02% (w/v) NaN₃, pH6.7 running buffer at 25° C. Fractions containing less than 2% oligomerswere pooled. The protein concentration was determined by measuring theoptical density (OD) at 280 nm, using the molar extinction coefficientcalculated on the basis of the amino acid sequence. Purity and molecularweight of the constructs were analyzed by SDS capillary electrophoresis(CE-SDS) in the presence and absence of a reducing agent following themanufacturer instructions (instrument Caliper LabChipGX, Perkin Elmer).Purified proteins were frozen in liquid N₂ and stored at −80° C.

TABLE 43 Yield, monomer content and purity by CE-SDS of the reducedaffinity 16D5 variants in the T cell bispecific format. Mutations toYield Purity by CE- Name reduce affinity [mg/L] Monomer [%] SDS [%] 16D5TCB G49S S93A 10.3 100 88 16D5 TCB G49S K53A 22.3 98.5 96 16D5 TCB W96Y15.2 98.7 92.5 16D5 TCB W96Y D52E 9.9 99.3 92.9 16D5 TCB Wild-type 5.496 91.6

All variants with reduced affinity could be produced in good quality.

Example 47 Binding of 36F2 TCB, 16D5 TCB and the Two 16D5 AffinityReduced Variants 16D5 W96Y/D52E TCB and 16D5 G49S/S93A TCB to HumanFolR1 Expressed on Hela Cells

The binding of 36F2 TCB, 16D5 TCB and the two 16D5 affinity reducedvariants 16D5 W96Y/D52E TCB and 16D5 G49S/S93A TCB to human FolR1 wasassessed on Hela cells. Briefly, cells were harvested, counted, checkedfor viability and resuspended at 2×10⁶ cells/ml in FACS buffer (100 μlPBS 0.1% BSA). 100 μl of cell suspension (containing 0.2×10⁶ cells) wasincubated in round-bottom 96-well plates for 30 min at 4° C. withdifferent concentrations of the bispecific antibodies (30 pM-500 nM).After two washing steps with cold PBS 0.1% BSA, samples werere-incubated for further 30 min at 4° C. with a FITC-conjugatedAffiniPure F(ab′)2 Fragment goat anti-human IgG Fcg Fragment Specificsecondary antibody (Jackson Immuno Research Lab PE #109-096-098). Afterwashing the samples twice with cold PBS 0.1% BSA samples werecentrifuged, resuspended in PBS 0.1% BSA and analyzed by FACS using aFACS CantoII (Software FACS Diva). Binding curves were obtained usingGraphPadPrism6 (FIG. 33).

Example 48 Production and Purification of Three T-Cell Bispecifics withIntermediate Affinity to Human and Cynomolgus FolR1: 14B1, 6E10, 2C7

Transient Transfection and Production

The intermediate affinity TCBs were transiently produced in HEK293 EBNAcells using a PEI mediated transfection procedure for the requiredvectors as described below. For transfection HEK293 EBNA cells arecultivated in suspension serum free in Excell culture medium containing6 mM L-Glutamine and 250 mg/l G418 culture medium. For the production in600 ml tubespin flask (max. working volume 400 mL) 600 million HEK293EBNA cells are seeded 24 hours before transfection. For transfectioncells are centrifuged for 5 min by 210×g, supernatant is replaced bypre-warmed 20 ml CD CHO medium. Expression vectors are mixed in 20 ml CDCHO medium to a final amount of 400 μg DNA. After addition of 1080 μlPEI solution (2.7 μg/ml) is vortexed for 15 s and subsequently incubatedfor 10 min at room temperature. Afterwards cells are mixed with theDNA/PEI solution, transferred to a 600 ml tubespin flask and incubatedfor 3 hours by 37° C. in an incubator with a 5% CO2 atmosphere. Afterincubation time 360 ml Excell+6 mM L-Glutamine+5 g/L Pepsoy+1.0 mM VPAmedium is added and cells are cultivated for 24 hours. One day aftertransfection 7% Feed 7 is added. After 7 days cultivation supernatant iscollected for purification by centrifugation for 20-30 min at 3600×g(Sigma 8K centrifuge), the solution is sterile filtered (0.22 μm filter)and sodium azide in a final concentration of 0.01% w/v is added, andkept at 4° C.

Purification

The intermediate affinity TCBs were purified in two steps using standardprocedures, such as protein A affinity purification (Äkta Explorer) andsize exclusion chromatography. The supernatant obtained from transientproduction was adjusted to pH 8.0 (using 2 M TRIS pH 8.0) and applied toHiTrap Protein A (GE Healthcare, column volume (cv)=5 ml) equilibratedwith 8 column volumes (cv) buffer A (20 mM sodium phosphate pH 7.5, 20mM sodium citrate). After washing with 10 cv of buffer A, the proteinwas eluted using a pH gradient to buffer B (20 mM sodium citrate pH 3.0,100 mM NaCl, 100 mM glycine) over 20 cv. Fractions containing theprotein of interest were pooled and the pH of the solution was gentlyadjusted to pH 6.0 (using 0.5 M Na₂HPO₄ pH 8.0). Samples wereconcentrated to 1 ml using ultra-concentrators (Amicon Ultra-15, 30.000MWCO, Millipore) and subsequently applied to a HiLoad™ 16/60 Superdex™200 preparative grade (GE Healthcare) equilibrated with 20 mM Histidine,pH 6.0, 140 mM NaCl, 0.01% Tween 20. The aggregate content of elutedfractions was analyzed by analytical size exclusion chromatography.Therefore, 30 μl of each fraction was applied to a TSKgel G3000 SW XLanalytical size-exclusion column (Toso h) equilibrated in 25 mM K₂HPO₄,125 mM NaCl, 200 mM L-arginine monohydrochloride, 0.02% (w/v) NaN₃, pH6.7 running buffer at 25° C. Fractions containing less than 2% oligomerswere pooled. The protein concentration was determined by measuring theoptical density (OD) at 280 nm, using the molar extinction coefficientcalculated on the basis of the amino acid sequence. Purity and molecularweight of the constructs were analyzed by SDS capillary electrophoresis(CE-SDS) in the presence and absence of a reducing agent following themanufacturer instructions (instrument Caliper LabChipGX, Perkin Elmer).Purified proteins were frozen in liquid N₂ and stored at −80° C.

TABLE 44 Yield, monomer content and purity by CE-SDS of the intermediateaffinity TCBs. Purity by CE- Name Yield [mg/L] Monomer [%] SDS [%] 6E10TCB 2.3 93 95 14B1 TCB 1.8 94 70 9C7 TCB 3.4 98 99

All intermediate affinity T cell bispecifics could be produced. Theyields are not high. The quality is good for 9C7 and acceptable for 14B1and 6E10.

Example 49 Binding of 16D5 HC/LC Variants to Human FolR1 Expressed onHT-29 Cells

The binding of 36F2 TCB, 16D5 TCB and various HC/LC variants (FIG.34A-E) of 16D5 to human FolR1 was assessed on HT-29 cells. Briefly,cells were harvested, counted, checked for viability and resuspended at2×10⁶ cells/ml in FACS buffer (100 μl PBS 0.1% BSA). 100 μl of cellsuspension (containing 0.2×10⁶ cells) was incubated in round-bottom96-well plates for 30 min at 4° C. with different concentrations of thebispecific antibodies (229 pM-500 nM). After two washing steps with coldPBS 0.1% BSA, samples were re-incubated for further 30 min at 4° C. witha PE-conjugated AffiniPure F(ab′)2 Fragment goat anti-human IgG FcgFragment Specific secondary antibody (Jackson Immuno Research Lab PE#109-116-170). After washing the samples twice with cold PBS 0.1% BSAthey were fixed with 1% PFA overnight. Afterwards samples werecentrifuged, resuspended in PBS 0.1% BSA and analyzed by FACS using aFACS CantoII (Software FACS Diva). Binding curves were obtained usingGraphPadPrism6 (FIG. 34A-E).

Example 50 Binding of Intermediate FolR1 Binders to Human and MouseFolR1 and FolR2

Cross-reactivity of the intermediate FolR1 binders (6E10 TCB, 14B1 TCBand 9C7 TCB), as well as 16D5 TCB and 36F2 TCB to human and mouse FolR1and FolR2 was assessed in a FACS binding assay on transfected HEK293Tcells.

Briefly, cells were harvested, counted, checked for viability andresuspended at 2×10⁶ cells/ml in FACS buffer (100 μl PBS 0.1% BSA). 100μl of cell suspension (containing 0.2×10⁶ cells) was incubated inround-bottom 96-well plates for 30 min at 4° C. with 100 nM of thebispecific antibodies. After two washing steps with cold PBS 0.1% BSA,samples were re-incubated for further 30 min at 4° C. with a Fluorescein(FITC) AffiniPure F(ab′)₂ Fragment Goat Anti-Human IgG, Fcγ FragmentSpecific secondary antibody (Jackson Immuno Research Lab PE#109-096-098). After washing the samples twice with cold PBS 0.1% BSAthey were fixed with 1% PFA overnight. Afterwards samples werecentrifuged, resuspended in PBS 0.1% BSA and analyzed by FACS using aFACS CantoII (Software FACS Diva). Graphs were obtained usingGraphPadPrism6 (FIG. 35A-D).

The results show that 36F2 TCB and 14B1 TCB are cross-reactive to mouseFolR1 and human and mouse FolR2. For 6E10 TCB a weak binding to humanFolR2 can be observed. 16D5 TCB and 9C7 TCB are specific for human FolR1and show no cross-reactivity to mouse FolR1 or human and mouse FolR2.

Example 51 Biochemical Characterization by Surface Plasmon Resonance of16D5 Reduced Affinity Variants and Additional Intermediate AffinityBinders in the T-Cell Bispecific Format

Binding of anti-FolR1 16D5 reduced affinity variants and additionalintermediate affinity binders in the bivalent T-cell bispecific formatto recombinant human, cynomolgus and murine folate receptor 1 (all as Fcfusions) was assessed by surface plasmon resonance (SPR). All SPRexperiments were performed on a Biacore T200 at 25° C. with HBS-EP asrunning buffer (0.01 M HEPES pH 7.4, 0.15 M NaCl, 3 mM EDTA, 0.005%Surfactant P20, Biacore, GE Healthcare). The molecules used for affinityand avidity determination are described in Table 45.

TABLE 45 Name, description and figure reference of the nine constructsused in SPR analysis. Figure Name Description reference 16D5 reducedaffinity 2 + 1 T-cell bispecific, inverted FIG. 1A variants format(common light chain) 16D5 TCB 16D5 G49S/S93A TCB 16D5 G49S/K53A TCB 16D5W96Y TCB 16D5 W96Y/D52E TCB Intermediate affinity 2 + 1 T-cellbispecific, inverted FIG. 1F binders format, crossfab 36F2 TCB 6E10 TCB14B1 TCB 9C7 TCB

Single Injections

First the anti-FolR1 TCBs were analyzed by single injections (Table 46)to characterize their crossreactivity (to human, murine and cyno FolR1)and specificity (to human FolR1, human FolR2, human FolR3). Recombinantbiotinylated monomeric Fc fusions of human, cynomolgus and murine FolateReceptor 1 (FolR1-Fc) or human Folate Receptor 2 and 3 (FolR2-Fc,FolR3-Fc) were directly coupled on a SA chip using the standard couplinginstruction (Biacore, Freiburg/Germany). The immobilization level wasabout 300-400 RU. The TCBs were injected for 60 seconds at aconcentration of 500 nM.

TABLE 46 Crossreactivity and specificity of 7 folate receptor 1 T cellbispecifics. + means binding, − means no binding, +/− means weakbinding. Binding to Binding to Binding to Binding to Binding to Clonename huFolR1 cyFolR1 muFolR1 huFolR2 huFolR3 16D5 TCB + + − − − 16D5 + +− − − G49S/S93A TCB 16D5 + + − − − W96Y/D52E TCB 36F2 TCB + + + +/− −6E10 TCB + + − − − 14B1 TCB + + + +/− − 9C7 TCB + + − − −

Avidity to Folate Receptor 1

The avidity of the interaction between the anti-FolR1 T cell bispecificsand the recombinant folate receptors was determined as described below(Table 47).

Recombinant biotinylated monomeric Fc fusions of human, cynomolgus andmurine Folate Receptor 1 (FolR1-Fc) were directly coupled on a SA chipusing the standard coupling instruction (Biacore, GE Healthcare). Theimmobilization level was about 200-300 RU. The anti-FolR1 T cellbispecifics were passed at a concentration range from 11.1 to 900 nM(for the 16D5 reduced affinity variants) or 0.2 to 500 nM (for theadditional intermediate affinity binders and 36F2) with a flow of 30μL/minutes through the flow cells over 180 seconds. The dissociation wasmonitored for 240 or 600 seconds. The chip surface was regenerated afterevery cycle using a double injection of 30 sec 10 mM Glycine-HCl pH 1.5.Bulk refractive index differences were corrected for by subtracting theresponse obtained on reference flow cell immobilized with recombinantbiotinylated murine IL2R Fc fusion (unrelated Fc fused receptor). Thebinding curves resulting from the bivalent binding of the T cellbispecifics were approximated to a 1:1 Langmuir binding (even though itis a 1:2 binding) and fitted with that model to get an apparent KDrepresenting the avidity of the bivalent binding. The apparent avidityconstants for the interactions were derived from the rate constants ofthe fitting using the Bia Evaluation software (GE Healthcare).

TABLE 47 Bivalent binding (avidity with apparent KD) of anti-FolR1T-cell bispecifics (TCB) on human, cyno and murine FolR1. ApparentAnalyte Ligand ka (1/Ms) kd (1/s) KD 16D5 TCB huFolR1 1.68E+05 4.33E−043 nM cyFolR1 2.08E+05 6.95E−04 3 nM 16D5 G49S/S93A huFolR1 1.49E+052.09E−03 10 nM  TCB cyFolR1 4.54E+05 7.84E−03 20 nM  16D5 G49S/K53AhuFolR1 1.32E+05 5.86E−03 40 nM  TCB cyFolR1 3.73E+05 2.56E−02 70 nM 16D5 W96Y TCB huFolR1 1.15E+05 1.44E−03 10 nM  cyFolR1 1.37E+05 1.68E−0310 nM  16D5 W96Y/D52E huFolR1 1.24E+05 1.40E−03 10 nM  TCB cyFolR15.17E+05 1.41E−02 30 nM  36F2 TCB huFolR1 1.12E+06 7.90E−03 7 nM cyFolR11.97E+06 1.10E−02 6 nM muFolR1 5.54E+05 1.47E−03 3 nM 6E10 TCB huFolR17.93E+06 8.74E−03 1 nM cyFolR1 5.56E+06 5.72E−03 1 nM 14B1 TCB huFolR11.12E+06 1.40E−03 1 nM cyFolR1 1.02E+06 1.66E−03 2 nM muFolR1 8.03E+068.20E−04 0.1 nM   9C7 TCB huFolR1 1.18E+06 1.42E−03 1 nM cyFolR14.98E+06 4.82E−03 1 nM

3. Affinity to Folate Receptor 1

The affinity of the interaction between the anti-FolR1 T cellbispecifics and the recombinant folate receptors was determined asdescribed below (Table 48).

For affinity measurement, direct coupling of around 12000 resonanceunits (RU) of the anti-human Fab specific antibody (Fab capture kit, GEHealthcare) was performed on a CM5 chip at pH 5.0 using the standardamine coupling kit (GE Healthcare). Anti-FolR1 T cell bispecifics werecaptured at 20 nM with a flow rate of 10 μl/min for 40 sec, thereference flow cell was left without capture. Dilution series (12.3 to3000 nM) of human, cyno or murine Folate Receptor 1 Fc fusion werepassed on all flow cells at 30 μl/min for 240 sec to record theassociation phase. The dissociation phase was monitored for 300 s andtriggered by switching from the sample solution to HBS-EP. The chipsurface was regenerated after every cycle using a double injection of 60sec 10 mM Glycine-HCl pH 2.1. Bulk refractive index differences werecorrected for by subtracting the response obtained on the reference flowcell 1. The affinity constants for the interactions were derived fromthe rate constants by fitting to a 1:1 Langmuir binding using the BiaEvaluation software (GE Healthcare). For low affinity kinetics withassociation and dissociation phases too fast to be fitted by the 1:1Langmuir binding model, the steady state analysis model was appliedusing the Bia Evaluation software (GE Healthcare). The steady stateanalysis gives the KD of the binding reaction at equilibrium.

TABLE 48 Monovalent binding (affinity) of anti-FolR1 T-cell bispecifics(TCB) on human, cyno and murine FolR1. Analyte Ligand ka (1/Ms) kd (1/s)KD 16D5 TCB huFolR1 1.22E+04 7.02E−04  57 nM cyFolR1 1.29E+04 1.71E−03 130 nM 16D5 G49S/ huFolR1 1.01E+04 8.37E−03  830 nM S93A TCB cyFolR12.05E+04 8.60E−03  420 nM 16D5 G49S/ huFolR1 9.17E+03 1.59E−02 1700 nMK53A TCB cyFolR1 1900 nM (steady state analysis) 16D5 W96Y huFolR11.11E+04 4.05E−03  370 nM TCB cyFolR1 1.17E+04 5.16E−03  440 nM 16D5huFolR1 1400 nM W96Y/ D52E TCB (steady state analysis) cyFolR1 5600 nM(steady state analysis) 36F2 TCB huFolR1 1400 nM (steady state analysis)cyFolR1 1500 nM (steady state analysis) muFolR1 3.50E+04 1.73E−02  490nM 6E10 TCB huFolR1 1200 nM (steady state analysis) cyFolR1 1500 nM(steady state analysis) 14B1 TCB huFolR1 6.16E+04 3.03E−02  490 nMcyFolR1 1200 nM (steady state analysis) muFolR1 7.03E+04 2.28E−03  30 nM9C7 TCB huFolR1  840 nM (steady state analysis) cyFolR1 1400 nM (steadystate analysis)

The mutations introduced into the 16D5 binders reduce its affinity tohuman and cynomolgus FolR1 as determined by surface plasmon resonance.The ranking with decreasing affinity is 16D5 WT (57 nM)>W96Y (6.5 foldlower)>G49S/S93A (14.5 fold lower)>W96Y/D52E (24.5 fold lower)>G49S/K53A(30 fold lower). The same ranking is visible in the avidity values,however the fold differences are smaller 16D5 WT (3 nM)>W96Y, G49S/S93A,W96Y/D52E (3 fold lower)>G49S/K53A (13 fold lower).

The intermediate affinity binders have following ranking in affinity16D5 (57 nM)>14B1 (8.5 fold lower)>9C7 (15 fold lower)>6E10 (21 foldlower)>36F2 (24.5 fold lower). These differences however disappear inthe avidity measurement 14B1, 9C7, 6E10 (1 nM)>16D5 (3 nM)>36F2 (7 nM).

16D5 W96Y/D52E TCB addresses the problems observed with previouscandidates. 16D5 W96Y/D52E TCB is based on the common light chain 16D5binder and has two point mutations on the heavy chain with respect tothe parental 16D5 binder. The W96Y mutation reduces the affinity of thebinder to FolR1 compared to the parental binder and the D52E mutationremoves a deamidation site and also contributes to the reduction inaffinity. 16D5 W96Y/D52E TCB binds to human and cynomolgus FolR1, butnot to murine FolR1. It is specific for FolR1 and does not bind torecombinant human FolR2 or human FolR3. The affinity (monovalentbinding) of 16D5 W96Y/D52E is around 1.4 μM for human FolR1 (24.5 foldlower than the parental 16D5 binder) and the avidity (bivalent binding)is around 10 nM (3 fold lower than the parental 16D5 binder).

Example 52 T-Cell Killing of Hela, SKov-3 and HT-29 Cells Induced byIntermediate FolR1 TCBs

T-cell killing mediated by intermediate FolR1 binders (6E10 TCB, 14B1TCB and 9C7 TCB), was assessed on Hela (high FolR1), SKov-3 (mediumFolR1) and HT-29 (low FolR1) cells. 16D5 TCB and 36F2 TCB were includedas benchmarks. Human PBMCs were used as effectors and the killing wasdetected at 24 h and 48 h of incubation with the bispecific antibodies.Briefly, target cells were harvested with Trypsin/EDTA, washed, andplated at a density of 25 000 cells/well using flat-bottom 96-wellplates. Cells were left to adhere overnight. Peripheral bloodmononuclear cells (PBMCs) were prepared by Histopaque densitycentrifugation of enriched lymphocyte preparations (buffy coats)obtained from healthy human donors. Fresh blood was diluted with sterilePBS and layered over Histopaque gradient (Sigma, #H8889). Aftercentrifugation (450×g, 30 minutes, room temperature), the plasma abovethe PBMC-containing interphase was discarded and PBMCs transferred in anew falcon tube subsequently filled with 50 ml of PBS. The mixture wascentrifuged (400×g, 10 minutes, room temperature), the supernatantdiscarded and the PBMC pellet washed twice with sterile PBS(centrifugation steps 350×g, 10 minutes). The resulting PBMC populationwas counted automatically (ViCell) and stored in RPMI1640 mediumcontaining 10% FCS and 1% L-alanyl-L-glutamine (Biochrom, K0302) at 37°C., 5% CO2 in cell incubator until further use (no longer than 24 h).For the killing assay, the antibody was added at the indicatedconcentrations (range of 0.01 pM-10 nM in triplicates). PBMCs were addedto target cells at final E:T ratio of 10:1. Target cell killing wasassessed after 24 h and 48 h of incubation at 37° C., 5% CO₂ byquantification of LDH released into cell supernatants byapoptotic/necrotic cells (LDH detection kit, Roche Applied Science, #11644 793 001). Maximal lysis of the target cells (=100%) was achieved byincubation of target cells with 1% Triton X-100. Minimal lysis (=0%)refers to target cells co-incubated with effector cells withoutbispecific construct.

The results show that tumor lysis induced by the intermediate FolR1binders (6E10 TCB, 14B1 TCB and 9C7 TCB) ranges between the one obtainedfor the high affinity 16D5 TCB and the low affinity 36F2 TCB (FIG.36A-F). Among the intermediate FolR1 binders, 14B1 TCB shows thestrongest killing as can be seen after 48 h of incubation (FIG. 36D-F).The EC50 values related to killing assays after 24 h and 48 h ofincubation, calculated using GraphPadPrism6, are given in Table 49 andTable 50.

TABLE 49 EC50 values (pM) for T-cell mediated killing of Hela, SKov-3and HT-29 cells induced by intermediate FolR1 TCBs after 24 h ofincubation. EC50 [pM] Antibody Hela SKov-3 HT-29 6E10 TCB 6.5 n.d. *n.d.14B1 TCB 8.5 30.1  *n.d. 9C7 TCB 2.8 741.4  *n.d. 16D5 TCB 2.2 1.5 *n.d.36F2 TCB 31.1 *n.d. *n.d. *not determined

TABLE 50 EC50 values (pM) for T-cell mediated killing of Hela, SKov-3and HT-29 cells induced by intermediate FolR1 TCBs after 48 h ofincubation. EC50 [pM] Antibody Hela SKov-3 HT-29 6E10 TCB 2.1 2164.0*n.d. 14B1 TCB 5.5 4.7 397.7 9C7 TCB 4.3 519.6 *n.d. 16D5 TCB 2.3 *n.d. 4.9 36F2 TCB 10.5 *n.d. n.d. *not determined

Example 53 T-Cell Killing of Hela, SKov-3 and HT-29 Cells Induced byAffinity Reduced 16D5 Variants

T-cell killing mediated by affinity reduced 16D5 variants(16D5-G49S/S93A TCB, 16D5-G49S/K53A TCB, 16D5 W96Y TCB, 16D5 W96Y/D52ETCB) was assessed on Hela (high FolR1), SKov-3 (medium FolR1) and HT-29(low FolR1) cells. 16D5 TCB and 36F2 TCB were included as benchmarks.The assay was performed as described above (Example 52).

The results show that tumor lysis induced by affinity reduced 16D5variants (16D5-G49S/S93A TCB, 16D5-G49S/K53A TCB, 16D5 W96Y TCB, 16D5W96Y/D52E TCB), ranges between the one obtained for the high affinity16D5 TCB and the low affinity 36F2 TCB. The EC50 values related tokilling assays after 24 h and 48 h of incubation, calculated usingGraphPadPrism6, are given in Table 51 and Table 52 (FIG. 37A-F).

TABLE 51 EC50 values (pM) for T-cell mediated killing ofFolR1-expressing Hela, SKov-3 and HT-29 cells induced by 16D5 TCB andits affinity reduced variants after 24 h of incubation. EC50 [pM]Antibody Hela SKov-3 HT-29 16D5 TCB 2.2 1.5 *n.d. 16D5- 2.3 430.4 *n.d.G49S/S93A TCB 16D5- 4.4 1701.9 *n.d. G49S/K53A TCB 16D5 W96Y 3.0 164.5*n.d. TCB 16D5 1.3 235.4 *n.d. W96Y/D52E TCB 36F2 TCB 31.1 *n.d. *n.d.*not determined

TABLE 52 EC50 values (pM) for T-cell mediated killing ofFolR1-expressing Hela, SKov-3 and HT-29 cells induced by 16D5 TCB andits affinity reduced variants after 48 h of incubation. EC50 [pM]Antibody Hela SKov-3 HT-29 16D5 TCB 2.3 0.1 4.9 16D5- 0.9 95.9 99.3G49S/S93A TCB 16D5- 0.5 950.4 1790.7 G49S/K53A TCB 16D5 W96Y 1.8 24.799.3 TCB 16D5 0.9 93.0 399.4 W96Y/D52E TCB 36F2 TCB 10.5 968.5 *n.d.*not determined

Thus, as with 36F2 FOLR1 TCB described above, the 16D5 W96Y/D52E TCBdifferentiates between high and low expressing cells which is of specialimportance to reduce toxicity as the cells of some normal, non-tumoroustissues express very low levels of FolR1 (approximately less than 1000copies per cell). Consistent with this observation, the resultsdiscussed in Example 54 below show that 16D5 W96Y/D52E TCB induces muchlower levels of T-cell-mediated killing of primary cells (FIG. 38A-F)compared to the parental 16D5 TCB. As such, 16D5 W96Y/D52E TCB mediatespotent killing of tumor tissues with high or medium FOLR1 expression,but not of normal tissues with low expression. 16D5 W96Y/D52E TCB in thebivalent 2+1 format comprises FolR1 binding moieties of relatively lowaffinity but it possesses an avidity effect which allows fordifferentiation between high and low FolR1 expressing cells. Becausetumor cells express FolR1 at high or intermediate levels, this TCBselectively binds to tumor cells and not normal, non-cancerous cellsthat express FolR1 at low levels or not at all. As an additionaladvantage over the 36F2 FOLR1 TCB described above, the 16D5 W96Y/D52ETCB binds specifically to FolR1 and not to FolR2 or FolR3, furtherenhancing its safety for in vivo treatment.

In addition to the above advantageous characteristics, the 16D5W96Y/D52E TCB in the bivalent 2+1 inverted format also has the advantagethat it does not require chemical cross linking or other hybridapproach. This makes it suitable for manufacture of a medicament totreat patients, for example patients having FolR1-positive canceroustumors. The 16D5 W96Y/D52E TCB in the bivalent 2+1 inverted format canbe produced using standard CHO processes with low aggregates. Further,the 16D5 W96Y/D52E TCB in the bivalent 2+1 comprises human and humanizedsequences making it superior to molecules that employ rat and murinepolypeptides that are highly immunogenic when administered to humans.Furthermore, the 16D5 W96Y/D52E TCB in the bivalent 2+1 format wasengineered to abolish FcgR binding and, as such, does not cause FcgRcrosslinking and infusion reactions, further enhancing its safety whenadministered to patients.

As demonstrated by the results described above, its head-to-tailgeometry make the 16D5 W96Y/D52E TCB in the bivalent 2+1 inverted formata highly potent molecule that induces absolute target cell killing. Itsbivalency enhance avidity and potency, but also allow fordifferentiation between high and low expressing cells. Its preferencefor high or medium target expressing cells due to its avidity affectreduce toxicity resulting from T cell mediated killing of normal cellsthat express FolR1 at low levels.

A further advantage of the 16D5 W96Y/D52E TCB in the bivalent 2+1 formatand other embodiments disclosed herein is that their clinicaldevelopment does not require the use of surrogate molecules as they bindto human and cynomous FolR1. As such, the molecules disclosed hereinrecognize a different epitope than antibodies to FolR1 previouslydescribed that do not recognize FolR1 from both species (see also FIG.41).

Example 54 T-Cell Killing of Primary Cells Induced by Affinity Reduced16D5 Variants and Intermediate FolR1 TCBs

T-cell killing mediated by affinity reduced 16D5 variants(16D5-G49S/S93A TCB, 16D5 W96Y/D52E TCB) and the intermediate FolR1binder 14B1 TCB was assessed on primary cells (Human Renal CorticalEpithelial Cells (HRCEpiC) (ScienCell Research Laboratories; Cat No4110) and Human Retinal Pigment Epithelial Cells (HRPEpiC) (ScienCellResearch Laboratories; Cat No 6540)). HT-29 cells (low FolR1) wereincluded as control cell line. 16D5 TCB and 36F2 TCB were included asbenchmarks and DP47 TCB served as non-binding control.

The assay was performed as described in Example 52, with a concentrationrange of the antibodies of 0.1 pM-100 nM (in triplicates).

When human primary cells are used as targets, the overall lysis is muchlower due to a lower expression rate of FolR1 on these cells (FIG.38A-F). For the high affinity FolR1 binder 16D5 TCB a T-cell mediatedlysis can be observed on both primary cell types used. As observedpreviously when tumor cell lines were used as targets, lysis induced bythe intermediate FolR1 binder 14B1 TCB and the affinity reduced 16D5variants (16D5-G49S/S93A TCB, 16D5 W96Y/D52E TCB), ranges between theone obtained for the high affinity 16D5 TCB and the low affinity 36F2TCB. The significantly reduced lysis of cells that express FolR1 at lowlevels is consistent with low off target activity and the affinityreduced 16D5 variants 16D5-G49S/S93A TCB and 16D5 W96Y/D52E TCB are,thus, expected to be well tolerated in vivo.

Example 55 Single Dose PK of FOLR1 TCB Constructs in Female NOG Mice

Female NOD/Shi-scid/IL-2Rγ null (NOG) mice at an average age of 8-10weeks at start of experiment (purchased from Taconic, SOPF facility)were maintained under specific-pathogen-free condition with daily cyclesof 12 h light/12 h darkness according to committed guidelines (GV-Solas;Felasa; TierschG). Experimental study protocol was reviewed and approvedby local government (ZH193/2014). After arrival animals were maintainedfor one week to get accustomed to new environment and for observation.Continuous health monitoring was carried out on regular basis.

A single dose pharmacokinetic study (SDPK) was performed to evaluateexposure of FOLR1 TCB constructs (36F2, 16D5, 16D5 G49S/S93A and 16D5W96Y/D52E). An i.v. bolus administration of 0.5 mg/kg was administeredto NOG mice and blood samples were taken at selected time points forpharmacokinetic evaluation. Mouse serum samples were analyzed by ELISA.Biotinylated a-huCD3-CDR (mAb<ID-mAb<CD3>>M-4.25.93-IgG-Bi), testsamples, Digoxygenin labelled a-huFc antibody (mAb<H-FCpan>M-R10Z8E9-IgG-Dig) and anti-Digoxygenin detection antibody (POD)were added stepwise to a 96-well streptavidin-coated microtiter plateand incubated after every step for 1 h at roomtemperature. The plate iswashed three times after each step to remove unbound substances.Finally, the peroxidase-bound complex is visualized by adding ABTSsubstrate solution to form a colored reaction product. The reactionproduct intensity, which is photometrically determined at 405 nm (withreference wavelength at 490 nm), is proportional to the analyteconcentration in the serum sample. The calibration range of the standardcurve for the constructs is was 0.078 to 5 ng/ml, where 1.5 ng/ml is thelower limit of quantification (LLOQ).

The SDPK study revealed an IgG-like PK-profile for the 16D5, 16D5W96Y/D52E and 16D5 G49S/S93A constructs (FIG. 39A-B). Because of that, aonce per weeks scheduling was chosen for the efficacy study (FIG. 40B).The half-life for 36F2 is lower as compared to the other clones. 36F2 isthe only out of the four molecules tested that is cross-reactive tomouse FOLR1, which might explain the lower half-life for this moleculeand indicates a TMDD (Target Mediated Drug Disposition).

Example 56 In Vivo Efficacy of FOLR1 TCB Constructs (16D5, 16D5G49S/S93A and 16D5 W96Y/D52E) after Human PBMC Transfer in Hela-BearingNOG Mice

The FOLR1 TCB constructs were tested in the FOLR1-expressing humancervical cancer cell line Hela, injected s.c. into PBMC engrafted NOGmice.

Hela cells were originally obtained from ATCC (CCL2) and after expansiondeposited in the Roche-Glycart internal cell bank. The tumor cell linewas routinely cultured in RPMI containing 10% FCS (Gibco) at 37° C. in awater-saturated atmosphere at 5% CO2. Passage 13 was used fortransplantation, at a viability >95%. 1×106 cells per animal wereinjected s.c. into the right flank of the animals in a total of 100 μlof RPMI cell culture medium (Gibco).

60 female NOG mice, age 8-10 weeks at start of the experiment (bred atTaconic, Denmark) were maintained under specific-pathogen-free conditionwith daily cycles of 12 h light/12 h darkness according to committedguidelines (GV-Solas; Felasa; TierschG). The experimental study protocolwas reviewed and approved by local government (ZH193/2014). Afterarrival, animals were maintained for one week to get accustomed to thenew environment and for observation. Continuous health monitoring wascarried out on a regular basis.

According to the study protocol (FIG. 40B), mice were injected s.c. onstudy day 0 with 1×106 Hela cells. At study day 30, when tumor reached asize of app. 150 mm3, human PBMC of a healthy donor were isolated viathe Ficoll method and 10×106 cells were injected i.v. into thetumor-bearing mice. Two days after (day 32), mice were randomized andequally distributed in six treatment groups (n=10) followed by i.v.injection with either 16D5 (0.5 mg/kg), 16D5 G49S/S93A (2.5 or 0.5mg/kg) and 16D5 W96Y/D52E (2.5 or 0.5 mg/kg). All treatments group wereinjected once weekly for three weeks in total. Mice were injected i.v.with 200 μl of the appropriate solution. The mice in the vehicle groupwere injected with PBS. To obtain the proper amount of TCB per 200 thestock solutions were diluted with PBS when necessary. Tumor growth wasmeasured once weekly using a caliper (FIG. 40C-E) and tumor volume wascalculated as followed:

Tv: (W2/2)×L (W: Width,L: Length)

The once weekly injection of the FOLR1 TCB constructs resulted insignificant tumor regression (FIG. 40C-E). The efficacy of 16D5 (0.5mg/kg) and 16D5 W96Y/D52E16D5 (0.5 mg/kg) was comparable, whereas 16D5G49S/S93A (0.5 mg/kg) showed slight less potency. The higher doses of2.5 mg/kg of 16D5 W96Y/D52E16D5 and 16D5 G49S/S93A didn't show increasedefficacy compared to 0.5 mg/kg doses. For PD read-outs, mice weresacrificed at study day 52, tumors were removed, weighted and singlecell suspensions were prepared through an enzymatic digestion withCollagenase V, Dispase II and DNAse for subsequent FACS-analysis.Explanted tumors of all treatment groups showed significant lower tumorweight at study termination as compared to vehicle control tumors (FIG.40F). Single cell suspensions from tumors where stained for huCD45 andhuCD3 and DAPI for dead cell exclusion and were analyzed at the BDFortessa. The FACS analysis revealed statistically higher numbers ofinfiltrated CD3-positive human T-cells in the tumor tissue upontreatment with 16D5 as well as 16D5 W96Y/D52E16D5 compared to vehiclecontrol tumors (FIG. 40C).

Example 57 Toxicity Study in Cynomolgus Monkey

A pharmacokinetic (PK), pharmacodynamic (PD) and tolerability study isperformed to investigate the tolerability, PK and PD effects of a singleintravenous dose of affinity reduced 16D5 variant TCBs (e.g.,16D5-G49S/S93A TCB, 16D5 W96Y/D52E TCB) in cynomolgus monkeys. In thisstudy, naïve cynomolgus monkeys, (1 male and 1 female monkey/group),receive a single intravenous dose of affinity reduced 16D5 variant TCBs,including 16D5 W96Y/D52E TCB, following a dose escalating protocol.Exemplary dose levels include 0.003, 0.03, and 0.09 mg/kg. Standardtoxicity parameters (clinical signs, body weights, hematology & clinicalchemistry) and the kinetics of T cell numbers and activation status inblood and the kinetics of cytokine release are assessed. Blood samplesare also taken for PK for a period of 28 days for the measurement ofaffinity reduced 16D5 variant TCBs, including 16D5 W96Y/D52E TCB, and ofanti-drug antibodies.

Amino Acid Sequences of Exemplary Embodiments

1) FolR Binders Useful in Common Light Chain Format, Variable HeavyChain

Description Sequence Seq ID No 16A3QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYYMHWVRQAPGQGLE  1WMGIINPSGGSTSYAQKFQGRVIMIRDTSTSTVYMELSSLRSEDTAVYYCARNYYAGVTPFDYWGQGTLVTVSS 18D3QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYYMHWVRQAPGQGLE  2WMGIINPSGGSTSYAQKFQGRVIMIRDTSTSTVYMELSSLRSEDTAVYYCARNYYTGGSSAFDYWGQGTLVTVS 15H7QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYYMHWVRQAPGQGLE  3WMGIINPSGGSTSYAQKFQGRVIMIRDTSTSTVYMELSSLRSEDTAVYYCARNYYLFSTSFDYWGQGTLVTVSS 15B6QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYYMHWVRQAPGQGLE  4WMGIINPSGGSTSYAQKFQGRVIMIRDTSTSTVYMELSSLRSEDTAVYYCARNYYIGIVPFDYWGQGTLVTVSS 21D1QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYYMHWVRQAPGQGLE  5WMGIINPSGGSTSYAQKFQGRVIMIRDTSTSTVYMELSSLRSEDTAVYYCARNYYVGVSPFDYWGQGTLVTVSS 16F12QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYYMHWVRQAPGQGLE  6WMGIINPSGGSTSYAQKFQGRVIMIRDTSTSTVYMELSSLRSEDTAVYYCARNFTVLRVPFDYWGQGTLVTVSS 15A1QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYYMHWVRQAPGQGLE  7WMGIINPSGGSTSYAQKFQGRVIMIRDTSTSTVYMELSSLRSEDTAVYYCARNYYIGVVTFDYWGQGTLVTVSS 15A1 CDR1 SYYMH  8 15A1 CDR2IINPSGGSTSYAQKFQG  9 15A1 CDR3 NYYIGVVTFDY 10 19E5QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYYMHWVRQAPGQGLE 11WMGIINPSGGSTSYAQKFQGRVIMIRDTSTSTVYMELSSLRSEDTAVYYCARGEWRRYTSFDYWGQGTLVTVSS 19E5_CDR1 SYYMH  8 19E5_CDR2IINPSGGSTSYAQKFQG  9 19E5_CDR3 GEWRRYTSFDY 12 19A4QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYYMHWVRQAPGQGLE 13WMGIINPSGGSTSYAQKFQGRVIMIRDTSTSTVYMELSSLRSEDTAVYYCARGGWIRWEHFDYWGQGTLVTVSS 19A4_CDR1 SYYMH  8 19A4_CDR2IINPSGGSTSYAQKFQG  9 19A4_CDR3 GGWIRWEHFDY 14 16D5EVQLVESGGGLVKPGGSLRLSCAASGFTFSNAWMSWVRQAPGKGLE 15WVGRIKSKTDGGITDYAAPVKGRFTISRDDSKNTLYLQMNSLKTEDTAVYYCTTPWEWSWYDYWGQGTLVTVSS 16D5_CDR1 NAWMS 16 16D5_CDR2RIKSKTDGGTTDYAAPVKG 17 16D5_CD3R PWEWSWYDY 18 15E12EVQLVESGGGLVKPGGSLRLSCAASGFTFSNAWMSWVRQAPGKGLE 19WVGRIKSKTDGGITDYAAPVKGRFTISRDDSKNTLYLQMNSLKTEDTAVYYCTTPWEWSYFDYWGQGTLVTVSS 15E12_CDR1 NAWMS 16 15E12_CDR2RIKSKTDGGTTDYAAPVKG 17 15E12_CDR3 PWEWSYFDY 20 21A5EVQLVESGGGLVKPGGSLRLSCAASGFTFSNAWMSWVRQAPGKGLE 21WVGRIKSKTDGGITDYAAPVKGRFTISRDDSKNTLYLQMNSLKTEDTAVYYCTTPWEWAWFDYWGQGTLVTVSS 21A5_CDR1 NAWMS 16 21A5_CDR2RIKSKTDGGTTDYAAPVKG 17 21A5_CDR3 PWEWAWFDY 22 21G8EVQLVESGGGLVKPGGSLRLSCAASGFTFSNAWMSWVRQAPGKGLE 23WVGRIKSKTDGGITDYAAPVKGRFTISRDDSKNTLYLQMNSLKTEDTAVYYCTTPWEWAYFDYWGQGTLVTVSS 21G8_CDR1 NAWMS 16 21G8_CDR2RIKSKTDGGTTDYAAPVKG 17 21G8_CDR3 PWEWAYFDY 24 19H3QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYYMHWVRQAPGQGLE 25WMGIINPSGGSTSYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARTGWSRWGYMDYWGQGTLVTVSS 19H3_CDR1 SYYMH  8 19H3_CDR2IINPSGGSTSYAQKFQG  9 19H3_CDR3 TGWSRWGYMDY 26 20G6QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYYMHWVRQAPGQGLE 27WMGIINPSGGSTSYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARGEWIRYYHFDYWGQGTLVTVSS 20G6_CDR1 SYYMH  8 20G6_CDR2IINPSGGSTSYAQKFQG  9 20G6_CDR3 GEWIRYYHFDY 28 20H7QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYYMHWVRQAPGQGLE 29WMGIINPSGGSTSYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARVGWYRWGYMDYWGQGTLVTVSS 20H7_CDR1 SYYMH  8 20H7_CDR2IINPSGGSTSYAQKFQG  9 20H7_CDR3 VGWYRWGYMDY 30

2) CD3 Binder Common Light Chain (CLC)

Description Sequence Seq ID No commonQAVVTQEPSLTVSPGGTVTLTCGSSTGAVTTSNYANWVQEKP 31 CD3 lightGQAFRGLIGGTNKRAPGTPARFSGSLLGGKAALTLSGAQPED chain (VL)EAEYYCALWYSNLWVFGGGTKLTVL common GSSTGAVTTSNYAN 32 CD3 light chain_CDR1common GTNKRAP 33 CD3 light chain_CDR2 common ALWYSNLWV 34 CD3 lightchain_CDR3 common QAVVTQEPSLTVSPGGTVTLTCGSSTGAVTTSNYANWVQEKP 35CD3 light GQAFRGLIGGTNKRAPGTPARFSGSLLGGKAALTLSGAQPED chainEAEYYCALWYSNLWVFGGGTKLTVLGQPKAAPSVTLFPPSSE (VLCL)ELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVA PTECS

3) CD3 Binder, Heavy Chain

Seq ID Description Sequence No CD3EVQLLESGGGLVQPGGSLRLSCAASGFTFSTYAMNWVRQAPGKG 36 variableLEWVSRIRSKYNNYATYYADSVKGRFTISRDDSKNTLYLQMNSL heavy chainRAEDTAVYYCVRHGNFGNSYVSWFAYWGQGTLVTVSS (VH) CD3 heavy TYAMN 37 chain(VH)_CDR1 CD3 heavy RIRSKYNNYATYYADSVKG 38 chain (VH)_CDR2 CD3 heavyHGNFGNSYVSWFAY 39 chain (VH)_CDR3 CD3 fullEVQLLESGGGLVQPGGSLRLSCAASGFTFSTYAMNWVRQAPGKG 40 heavy chainLEWVSRIRSKYNNYATYYADSVKGRFTISRDDSKNTLYLQMNSL (VHCH1)_ RAEDTAVYYCVRHGNFGNSYVSWFAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVD KKVEPKSC CD3ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSG 84 constantALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHK heavy chain PSNTKVDKKVEPKSCCH1

4) FolR Binders Useful for Crossfab Format

Seq ID Description Sequence No 11F8_VHQVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLE 41WMGGIIPIFGTANYAQKFQGRVTITADKSTSTAYMELSSLRSEDTAVYYCARAVFYRAWYSFDYWGQGTTVTVSS 11F8_VH_CDR1 SYAIS 42 11F8_VH_CDR2GIIPIFGTANYAQKFQG 43 11F8_VH_CDR3 AVFYRAWYSFDY 44 11F8_VLDIQMTQSPSTLSASVGDRVTITCRASQSISSWLAWYQQKPGKAPKL 45LIYDASSLESGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCQQYT SPPPTFGQGTKVEIK11F8_VL_CDR1 RASQSISSWLA 46 11F8_VL_CDR2 DASSLES 47 11F8_VL_CDR3QQYTSPPPT 48 36F2_VH QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYYMHWVRQAPGQGLE 49WMGIINPSGGSTSYAQKFQGRVIMTHDTSTSTVYMELSSLRSEDTAVYYCARSFFTGFHLDYWGQGTLVTVSS 36F2_VH_CDR1 SYYMH  8 36F2_VH_CDR2IINPSGGSTSYAQKFQG  9 36F2_VH_CDR3 SFFTGFHLDY 50 36F2_VLEIVLIQSPGILSLSPGERATLSCRASQSVSSSYLAWYQQKPGQAPR 51LLIYGASSRATGIPDRFSGSGSGTDFILTISRLEPEDFAVYYCQQY TNEHYYTFGQGTKVEIK36F2_VL_CDR1 RASQSVSSSYLA 52 36F2_VL_CDR2 GASSRAT 53 36F2_VL_CDR3QQYTNEHYYT 54 9D11_VH QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYYMHWVRQAPGQGLE 55WMGIINPSGGPTSYAQKFQGRVIMIRDTSTSTVYMELSSLRSEDTA VYYCARGDFAWLDYWGQGTLVTVSS9D11_VH_CDR1 SYYMH  8 9D11_VH_CDR2 IINPSGGPTSYAQKFQG 56 9D11_VH_CDR3GDFAWLDY 57 9D11_VL DIVMTQSPLSLPVTPGEPASISCRSSQSLLHSNGYNYLDWYLQKPG 58QSPQLLIYLGSNRASGVPDRFSGSGSGTDFTLKISRVEAEDVGVYY CMQASIMNRTFGQGTKVEIK9D11_VL_CDR1 RSSQSLLHSNGYNYLD 59 9D11_VL_CDR2 LGSNRAS 60 9D11_VL_CDR3MQASIMNRT 61 9D11_VL DIVMTQSPLSLPVTPGEPASISCRSSQSLLHSNGYNYLDWYLQKPG 62N95S QSPQLLIYLGSNRASGVPDRFSGSGSGTDFTLKISRVEAEDVGVYY CMQASIMSRTFGQGTKVEIK9D11_VL MQASIMSRT 63 N95S_CDR3 9D11_VLDIVMTQSPLSLPVTPGEPASISCRSSQSLLHSNGYNYLDWYLQKPG 64 N95QQSPQLLIYLGSNRASGVPDRFSGSGSGTDFTLKISRVEAEDVGVYY CMQASIMQRTFGQGTKVEIK9D11_VL MQASIMQRT 65 N95Q_CDR3 9D11_VLDIVMTQSPLSLPVTPGEPASISCRSSQSLLHSNGYNYLDWYLQKPG 66 T97AQSPQLLIYLGSNRASGVPDRFSGSGSGTDFTLKISRVEAEDVGVYY CMQASIMNRAFGQGTKVEIK9D11_VL MQASIMNRA 67 T97A 9D11_VLDIVMTQSPLSLPVTPGEPASISCRSSQSLLHSNGYNYLDWYLQKPG 68 T97NQSPQLLIYLGSNRASGVPDRFSGSGSGTDFTLKISRVEAEDVGVYY CMQASIMNRNFGQGTKVEIK9D11_VL MQASIMNRN 69 T97N_CDR3 5D9_VHQVQLVQSGAEVKKPGASVKVSCKASGYTFTSYYMHWVRQAPGQGLE 70WMGIINPSGGSTSYAQKFQGRVIMIRDTSTSTVYMELSSLRSEDTA VYYCARSYIDMDYWGQGTLVTVSS5D9_VH_CDR1 SYYMH  8 5D9_VH_CDR2 IINPSGGSTSYAQKFQG  9 5D9_VH_CDR3SYIDMDY 71 5D9_VL EIVLIQSPGILSLSPGERATLSCRASQSVSSSYLAWYQQKPGQAPR 72LLIYGASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQD NWSPTFGQGTKVEIK5D9_VL_CDR1 RASQSVSSSYLA 52 5D9_VL_CDR2 GASSRAT 53 5D9_VL_CDR3 QQDNWSPT73 6B6_VH QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYYMHWVRQAPGQGLE 74WMGIINPSGGSTSYAQKFQGRVIMIRDTSTSTVYMELSSLRSEDTA VYYCARSYVDMDYWGQGTLVTVSS6B6_VH_CDR1 SYYMH  8 6B6_VH_CDR2 IINPSGGSTSYAQKFQG  9 6B6_VH_CDR3SYVDMDY 75 6B6_VL EIVLIQSPGILSLSPGERATLSCRASQSVSSSYLAWYQQKPGQAPR 76LLIYGASSRATGIPDRFSGSGSGTDFILTISRLEPEDFAVYYCQQD IWSPTFGQGTKVEIK6B6_VL_CDR1 RASQSVSSSYLA 52 6B6_VL_CDR2 GASSRAT 53 6B6_VL_CDR3 QQDIWSPT77 14E4_VH EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLE 78WVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKDSSYVEWYAFDYWGQGTLVTVSS 14E4_VH_CDR1 SYAMS 79 14E4_VH_CDR2AISGSGGSTYYADSVKG 80 14E4_VH_CDR3 DSSYVEWYAFDY 81 14E4_VLEIVLIQSPGILSLSPGERATLSCRASQSVSSSYLAWYQQKPGQAPR 82LLIYGASSRATGIPDRFSGSGSGTDSTLTISRLEPEDFAVYYCQQP TSSPITFG QGTKVEIK14E4_VL_CDR1 RASQSVSSSYLA 52 14E4_VL_CDR2 GASSRAT 53 14E4_VL_CDR3QQPTSSPIT 83

5) CD3 Binder Useful in Crossfab Format

Description Sequence Seq ID No CD3 heavyEVQLLESGGGLVQPGGSLRLSCAASGFTFSTYAMNWVRQAPG 36 chain (VH)KGLEWVSRIRSKYNNYATYYADSVKGRFTISRDDSKNTLYLQMNSLRAEDTAVYYCVRHGNFGNSYVSWFAYWGQGTLVTVSS CD3 heavy TYAMN 37 chain(VH)_CDR1 CD3 heavy RIRSKYNNYATYYADSVKG 38 chain (VH)_CDR2 CD3 heavyHGNFGNSYVSWFAY 39 chain (VH)_CDR3 CD3 lightQAVVTQEPSLTVSPGGTVTLTCGSSTGAVTTSNYANWVQEKP 31 chain (VL)GQAFRGLIGGTNKRAPGTPARFSGSLLGGKAALTLSGAQPED EAEYYCALWYSNLWVFGGGTKLTVLCD3 light GSSTGAVTTSNYAN 32 chain_CDR1 CD3 light GTNKRAP 33 chain_CDR2CD3 light ALWYSNLWV 34 chain_CDR3 pETR12940:QAVVTQEPSLTVSPGGTVTLTCGSSTGAVTTSNYANWVQEKP 86 crossedGQAFRGLIGGTNKRAPGTPARFSGSLLGGKAALTLSGAQPED commonEAEYYCALWYSNLWVFGGGTKLTVLSSASTKGPSVFPLAPSS CD3 lightKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVL chainQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVE (VLCH1) PKSC CrossedEVQLLESGGGLVQPGGSLRLSCAASGFTFSTYAMNWVRQAPG 87 CD3 heavyKGLEWVSRIRSKYNNYATYYADSVKGRFTISRDDSKNTLYLQ chainMNSLRAEDTAVYYCVRHGNFGNSYVSWFAYWGQGTLVTVSSA (VHCκ);SVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKV e.g. inDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVY pCON1057ACEVTHQGLSSPVTKSFNRGEC CD3- ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWN85 CH1 SGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICN VNHKPSNTKVDKKVEPKSCCD3- VAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVD 88 ckappaNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYA CEVTHQGLSSPVTKSFNRGEC

6)—Exemplary Amino Acid Sequences of CD3-FolR Bispecific Antibodies 2+1Inverted Crossmab Format

Description Sequence Seq ID No VHCH1 QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYYMHWVRQAPGQGLE  94 [9D11]_VHCLWMGIINPSGGPTSYAQKFQGRVIMIRDTSTSTVYMELSSLRSEDTA [CD3]_VYYCARGDFAWLDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGG Fcknob_TAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSS PGLALAVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDGGGGSGGGG pCON1057SEVQLLESGGGLVQPGGSLRLSCAASGFTFSTYAMNWVRQAPGKGLEWVSRIRSKYNNYATYYADSVKGRFTISRDDSKNTLYLQMNSLRAEDTAVYYCVRHGNFGNSYVSWFAYWGQGTLVTVSSASVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGECDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVICVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 9D11_Fchole_QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYYMHWVRQAPGQGLE  95 PGLALA_WMGIINPSGGPTSYAQKFQGRVIMIRDTSTSTVYMELSSLRSEDTA HYRFVYYCARGDFAWLDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRIPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 9D11_LCDIVMTQSPLSLPVTPGEPASISCRSSQSLLHSNGYNYLDWYLQKPG   96 pCON1063QSPQLLIYLGSNRASGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCMQASIMNRTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC VLCH1 QAVVTQEPSLTVSPGGTVTLTCGSSTGAVTTSNYANWVQEKPGQAF  86 [CD3]RGLIGGINKRAPGTPARFSGSLLGGKAALTLSGAQPEDEAEYYCAL pETR12940WYSNLWVFGGGTKLTVLSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSC CH1ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGAL 307TSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNT KVDKKVEPKSCD VHCH1 QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYYMHWVRQAPGQGLE 308 [36F2]_VHCLWMGIINPSGGSTSYAQKFQGRVTMTHDTSTSTVYMELSSLRSEDTA [CD3]_VYYCARSFFTGFHLDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTS Fcknob_GGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSL PGLALASSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDGGGGSGG pCON1056GGSEVQLLESGGGLVQPGGSLRLSCAASGFTFSTYAMNWVRQAPGKGLEWVSRIRSKYNNYATYYADSVKGRFTISRDDSKNTLYLQMNSLRAEDTAVYYCVRHGNFGNSYVSWFAYWGQGTLVTVSSASVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGECDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVICVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKITPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSP GK 36F2-FcQVQLVQSGAEVKKPGASVKVSCKASGYTFTSYYMHWVRQAPGQGLE 309 holeWMGIINPSGGSTSYAQKFQGRVTMTHDTSTSTVYMELSSLRSEDTA PGLALAVYYCARSFFTGFHLDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTS pCON1050GGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRIPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 36F2 LCEIVLIQSPGILSLSPGERATLSCRASQSVSSSYLAWYQQKPGQAPR 310 pCON1062LLIYGASSRATGIPDRFSGSGSGTDFILTISRLEPEDFAVYYCQQYTNEHYYTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHXGLSSPVTKSFNRGEC CD3QAVVTQEPSLTVSPGGTVTLTCGSSTGAVTTSNYANWVQEKPGQAF  86 VLCH1RGLIGGINKRAPGTPARFSGSLLGGKAALTLSGAQPEDEAEYYCAL pETR12940WYSNLWVFGGGTKLTVLSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSC

7) Exemplary Amino Acid Sequences of CD3-FolR Bispecific Antibodies withCommon Light Chain

VHCH1 EVQLVESGGGLVKPGGSLRLSCAASGFTFSNAWMSWVRQAPGKGLEWVG  89 [16D5]_RIKSKTDGGTTDYAAPVKGRFTISRDDSKNTLYLQMNSLKTEDTAVYYC VHCH1TTPWEWSWYDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGC [CD3]_LVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSL FcknobGTQTYICNVNHKPSNTKVDKKVEPKSCDGGGGSGGGGSEVQLLESGGGL pCON999VQPGGSLRLSCAASGFTFSTYAMNWVRQAPGKGLEWVSRIRSKYNNYATYYADSVKGRFTISRDDSKNTLYLQMNSLRAEDTAVYYCVRHGNFGNSYVSWFAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLS PGK VHCH1EVQLVESGGGLVKPGGSLRLSCAASGFTFSNAWMSWVRQAPGKGLEWVG  90 [16D5]_RIKSKTDGGTTDYAAPVKGRFTISRDDSKNTLYLQMNSLKTEDTAVYYC FcholeTTPWEWSWYDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGC pCON983LVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKITPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNRFTQ KSLSLSPGK CD3_commonQAVVTQEPSLIVSPGGIVTLICGSSTGAVITSNYANWVQEKPGQAFRGL  35 lightIGGINKRAPGTPARFSGSLLGGKAALTLSGAQPEDEAEYYCALWYSNLW chainVFGGGTKLTVLGQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAV pETR13197TVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSC QVTHEGSTVEKTVAPTECSVHCH1  EVQLLESGGGLVQPGGSLRLSCAASGFTFSTYAMNWVRQAPGKGLEWVS  91 [CD3]_RIRSKYNNYATYYADSVKGRFTISRDDSKNTLYLQMNSLRAEDTAVYYC VHCH1VRHGNFGNSYVSWFAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGT [16D5]_AALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTV Fcknob_PSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDGGGGSGGGGSEVQLVE PGLALASGGGLVKPGGSLRLSCAASGFTFSNAWMSWVRQAPGKGLEWVGRIKSKT pETR13932DGGTTDYAAPVKGRFTISRDDSKNTLYLQMNSLKTEDTAVYYCTTPWEWSWYDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLS PGK CD3_Fcknob_EVQLLESGGGLVQPGGSLRLSCAASGFTFSTYAMNWVRQAPGKGLEWVS  92 PGLALARIRSKYNNYATYYADSVKGRFTISRDDSKNTLYLQMNSLRAEDTAVYYC pETR13917VRHGNFGNSYVSWFAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALH NHYTQKSLSLSPGKFc_hole_ DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHE  93 PGLALA_DPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKE HYRFYKCKVSNKALPAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSC pETR10755AVKGFYPSDIAVEWESNGQPENNYKITPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNRFTQKSLSLSPGK VHCL[CD3]_EVQLLESGGGLVQPGGSLRLSCAASGFTFSTYAMNWVRQAPGKGLEWVS  98 Fcknob_RIRSKYNNYATYYADSVKGRFTISRDDSKNTLYLQMNSLRAEDTAVYYC PGLALAVRHGNFGNSYVSWFAYWGQGTLVTVSSASVAAPSVFIFPPSDEQLKSGT pETR13378ASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGECDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMH EALHNHYTQKSLSLSPGK16D5 EVQLVESGGGLVKPGGSLRLSCAASGFTFSNAWMSWVRQAPGKGLEWVG  99 inverted RIKSKTDGGTTDYAAPVKGRFTISRDDSKNTLYLQMNSLKTEDTAVYYC 2 + 1 with TTPWEWSWYDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGC N100A in LVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSL CDR H3GTQTYICNVNHKPSNTKVDKKVEPKSCDGGGGSGGGGSEVQLLESGGGL pETR14096VQPGGSLRLSCAASGFTFSTYAMNWVRQAPGKGLEWVSRIRSKYNNYATYYADSVKGRFTISRDDSKNTLYLQMNSLRAEDTAVYYCVRHGNFGASYVSWFAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLS PGK 16D5EVQLVESGGGLVKPGGSLRLSCAASGFTFSNAWMSWVRQAPGKGLEWVG 100 inverted RIKSKTDGGTTDYAAPVKGRFTISRDDSKNTLYLQMNSLKTEDTAVYYC 2 + 1 with TTPWEWSWYDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGC S100aA in LVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSL CDR H3GTQTYICNVNHKPSNTKVDKKVEPKSCDGGGGSGGGGSEVQLLESGGGL pETR14097VQPGGSLRLSCAASGFTFSTYAMNWVRQAPGKGLEWVSRIRSKYNNYATYYADSVKGRFTISRDDSKNTLYLQMNSLRAEDTAVYYCVRHGNFGNAYVSWFAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLS PGK CD3 lightQAVVTQEPSLIVSPGGIVTLICGSSTGAVITSNYANWVQEKPGQAFRGL 101 chain fusedIGGINKRAPGTPARFSGSLLGGKAALTLSGAQPEDEAEYYCALWYSNLW to CH1;VFGGGTKLTVLSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEP Fc_PGLALA;VTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNV pETR13862NHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 16D5 VHEVQLVESGGGLVKPGGSLRLSCAASGFTFSNAWMSWVRQAPGKGLEWVG 102 fused toRIKSKTDGGTTDYAAPVKGRFTISRDDSKNTLYLQMNSLKTEDTAVYYC constantTTPWEWSWYDYWGQGTLVTVSSASVAAPSVFIFPPSDEQLKSGTASVVC kappa chain;LLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSK pETR13859ADYEKHKVYACEVTHQGLSSPVTKSFNRGEC CD3 VHEVQLLESGGGLVQPGGSLRLSCAASGFTFSTYAMNWVRQAPGKGLEWVS 103 fused toRIRSKYNNYATYYADSVKGRFTISRDDSKNTLYLQMNSLRAEDTAVYYC constantVRHGNFGNSYVSWFAYWGQGTLVTVSSASPKAAPSVTLFPPSSEELQAN lambdaKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSY chain;LSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS pETR13860 IGHV1-QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYYMHWVRQAPGQGLEWMG 104 46*01IINPSGGSTSYAQKFQGRVIMIRDTSTSTVYMELSSLRSEDTAVYYCAR (X92343),GGSGGSFDYWGQGTLVTVSS plus JH4 element IGHV1-QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWMG 105 69*06GIIPIFGTANYAQKFQGRVTITADKSTSTAYMELSSLRSEDTAVYYCAR (L22583),GGSGGSMDAWGQGTTVTVSS plus JH6 element IGHV3-EVQLVESGGGLVKPGGSLRLSCAASGFTFSNAWMSWVRQAPGKGLEWVG 106 15*01RIKSKTDGGTTDYAAPVKGRFTISRDDSKNTLYLQMNSLKTEDTAVYYC (X92216),TTGGSGGSFDYWGQGTLVTVSS plus JH4 element IGHV3-EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVS 107 23*01AISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAK (M99660),GGSGGSFDYWGQGTLVTVSS plus JH4 element IGHV4-QVQLQESGPGLVKPSETLSLICTVSGGSISSYYWSWIRQPPGKGLEWIG 108 59*01YIYYSGSTNYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARG (AB019438),GSGGSFDYWGQGTLVTVSS plus JH4 element IGHV5-EVQLVQSGAEVKKPGESLKISCKGSGYSFTSYWIGWVRQMPGKGLEWMG 109 51*01IIYPGDSDTRYSPSFQGQVTISADKSISTAYLQWSSLKASDTAMYYCAR (M99686),GGSGGSFDYWGQGTLVTVSS plus JH4 element CD3 specificQTVVTQEPSLTVSPGGTVTLTCGSSTGAVTTSNYANWVQEKPGQAFRGL 110 antibodyIGGINKRAPGTPARFSGSLLGGKAALTLSGAQPEDEAEYYCALWYSNLW based on VFGGGTKLTVLhumanized CH2527 light chain hVK1-39DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIY 111 (JK4 J-AASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPLTF element) GGGTKVEIKVL7_46-13 QAVVTQEPSLTVSPGGTVTLTCGSSTGAVTTSNYANWVQEKPGQAFRGL 112(humanized IGGINKRAPGTPARFSGSLLGGKAALTLSGAQPEDEAEYYCALWYSNLW anti-CD3VFGGGTKLTVL antibody light chain)

8) Exemplary 16D5 Variants with Reduced Affinity

a. Exemplary Light Chain Variants with Reduced Affinity

Seq ID Name Sequence No K53A QTVVTQEPSLTVSPGGTVTLTC GSSTGAVTT 113 aaSNYAN WVQQKPGQAPRGLIG G TNARAP GTPA RFSGSLLGGKAALTLSGVQPEDEAEYYC ALWYSNLWV FGGGTKLTVL K53A_VL_ GSSTGAVTTSNYAN  32 CDR1 K53A_VL_ GTNARAP 311CDR2 K53A_VL_ ALWYSNLWV  34 CDR3 S93A QTVVTQEPSLTVSPGGTVTLTC GSSTGAVTT114 aa SNYAN WVQQKPGQAPRGLIG G TNKRAP GTPA RFSGSLLGGKAALTLSGVQPEDEAEYYCALW YANLWV FGGGTKLTVL S93A_VL_ GSSTGAVTTSNYAN  32 CDR1 S93A_VL_ GTNKRAP 33 CDR2 S93A_VL_ ALWYANLWV 312 CDR3

b. Exemplary Heavy Chain Variants with Reduced Affinity

Seq Name Sequence ID No S35H EVQLVESGGGLVKPGGSLRLSCAASGETFS 115 aa NAWMHWVRQAPGKGLEWVG RIKSKTDGGIT DYAAPVKG RFTISRDDSKNTLYLQMNSLKT EDTAVYYCTTPWEWSWYDY WGQGTLVTVSS AS S35H_VH_ NAWMH 313 CDR1 S35H_VH_RIKSKTDGGTTDYAAPVKG  17 CDR2 S35H_VH_ PWEWSWYDY  18 CDR3 G49SEVQLVESGGGLVKPGGSLRLSCAASGETFS 116 aa NAWMS WVRQAPGKGLEWVS RIKSKTDGGTTDYAAPVKG RFTISRDDSKNTLYLQMNSLKT EDTAVYYCTT PWEWSWYDY WGQGTLVTVSS ASG49S_VH_ NAWMS  16 CDR1 G49S_VH_ RIKSKTDGGTTDYAAPVKG  17 CDR2 G49S_VH_PWEWSWYDY  18 CDR3 R50S EVQLVESGGGLVKPGGSLRLSCAASGETFS 117 aa NAWMSWVRQAPGKGLEWVG SIKSKTDGGTT DYAAPVKG RFTISRDDSKNTLYLQMNSLKT EDTAVYYCTTPWEWSWYDY WGQGTLVTVSS AS R50S_VH_ NAWMS  16 CDR1 R50S_VH_SIKSKTDGGTTDYAAPVKG 314 CDR2 R50S_VH_ PWEWSWYDY  18 CDR3 W96YEVQLVESGGGLVKPGGSLRLSCAASGFTFS 118 aa NAWMS WVRQAPGKGLEWVG RIKSKTDGGTTDYAAPVKG RFTISRDDSKNTLYLQMNSLKT EDTAVYYCTTPYEW SWYDY WGQGTLVTVSS ASW96Y_VH_ NAWMS  16 CDR1 W96Y_VH_ RIKSKTDGGTTDYAAPVKG  17 CDR2 W96Y_VH_PYEWSWYDY 315 CDR3 W98Y EVQLVESGGGLVKPGGSLRLSCAASGFTFS 119 aa NAWMSWVRQAPGKGLEWVG RIKSKTDGGTT DYAAPVKG RFTISRDDSKNTLYLQMNSLKT EDTAVYYCTTPWEYSWYDY WGQGTLVTVSS AS W98Y_VH_ NAWMS  16 CDR1 W98Y_VH_RIKSKTDGGTTDYAAPVKG  17 CDR2 W98Y_VH_ PWEYSWYDY 232 CDR3

9) Additional Exemplary Embodiments Generated from a Phage DisplayLibrary (CDRs Underlined)

Name Sequence Seq ID No 90D7 QVQLVQSGAEVKKPGASVKVSCKASGYTF 120 aa TSYYMH WVRQAPGQGLEWMG IINPSGGST SYAQKFQG RVTMTRDTSTSTVYMELSSLRSEDTAVYYCAR NYTIVVSPFDY WGQGTLV TVSSAS 90D7_VH_ SYYMH   8 CDR1 90D7_VH_IINPSGGSTSYAQKFQG   9 CDR2 90D7_VH_ NYTIVVSPFDY 233 CDR3 90C1QVQLVQSGAEVKKPGASVKVSCKASGYTF 121 aa T SYYMH WVRQAPGQGLEWMG IINPSGGSTSYAQKFQG RVTMTRDTSTSTVYMELSSLR SEDTAVYYCAR NYFIGSVAMDY WGQGTLV TVSSAS90C1_VH_ SYYMH   8 CDR1 90C1_VH_ IINPSGGSTSYAQKFQG   9 CDR2 90C1_VH_NYFIGSVAMDY 234 CDR3 5E8 VH QVQLVQSGAEVKKPGASVKVSCKASGYTF 122 aa T SYYMHWVRQAPGQGLEWMG IINPSGGST SYAQKFQG RVTMTRDTSTSTVYMELSSLR SEDTAVYYCARGLTYSMDY WGQGTLVTVS SAS 5E8_VH_ SYYMH   8 CDR1 5E8_VH_ IINPSGGSTSYAQKFQG  9 CDR2 5E8_VH_ GLTYSMDY 235 CDR3 5E8 VL DIVMTQSPLSLPVTPGEPASISC RSSQSL123 aa LHSNGYNYLD WYLQKPGQSPQLLIY LGSN RAS GVPDRFSGSGSGTDFTLKISRVEAEDVGVYYC MQALQIPNT FGQGTKVEIKRT 5E8_VL_ RSSQSLLHSNGYNYLD  59 CDR1 5E8_VL_LGSNRAS  60 CDR2 5E8_VL_ MQALQIPNT 236 CDR3 12A4 VHEVQLLESGGGLVQPGGSLRLSCAASGETF 124 aa S SYAMS WVRQAPGKGLEWVS AISGSGGSTYYADSVKG RFTISRDNSKNTLYLQMNSLR AEDTAVYYCAK YAYALDY WGQGTLVTVSS AS12A4_VH_ SYAMS  79 CDR1 12A4_VH_ AISGSGGSTYYADSVKG  80 CDR2 12A4_VH_YAYALDY 237 CDR3 12A4 VL EIVLTQSPGTLSLSPGERATLSC RASQSV 125 aa SSSYLAWYQQKPGQAPRLLIY GASSRAT G IPDRFSGSGSGTDFTLTISRLEPEDFAVY YC QQHGSSSTFGQGTKVEIKRT 12A4_VL_ RASQSVSSSYLA  52 CDR1 12A4_VL_ GASSRAT  53 CDR212A4_VL_ QQHGSSST 238 CDR3 7A3 VH QVQLVQSGAEVKKPGASVKVSCKASGYTF 126 aa TSYYMH WVRQAPGQGLEWMG IINPSGGST SYAQKFQG RVTMTRDTSTSTVYMELSSLRSEDTAVYYCAR GDFSAGRLMDY WGQGTLV TVSSAS 7A3_VH_ SYYMH   8 CDR1 7A3_VH_IINPSGGSTSYAQKFQG   9 CDR2 7A3_VH_ GDFSAGRLMDY 239 CDR3 7A3 VLDIVMTQSPLSLPVTPGEPASISC RSSQSL 127 aa LHSNGYNYLD WYLQKPGQSPQLLIY LGSNRAS GVPDRFSGSGSGTDFTLKISRVEAED VGVYYC MQALQTPPIT FGQGTKVEIKRT 7A3_VL_RSSQSLLHSNGYNYLD  59 CDR1 7A3_VL_ LGSNRAS  60 CDR2 7A3_VL_ MQALQTPPIT240 CDR3 6E10 VH QVQLVQSGAEVKKPGASVKVSCKASGYTF 128 aa T SYYMHWVRQAPGQGLEWMG IINPSGGST SYAQKFQG RVTMTRDTSTSTVYMELSSLR SEDTAVYYCARGDYNAFDY WGHGTLVTVS SAS 6E10 VH_ SYYMH   8 CDR1 6E10 VH_IINPSGGSTSYAQKFQG   9 CDR2 6E10 VH_ GDYNAFDY 241 CDR3 6E10 VLDIVMTQSPLSLPVTPGEPASISC RSSQSL 129 aa LHSNGYNYLD WYLQKPGQSPQLLIY LGSNRAS GVPDRFSGSGSGTDFTLKISRVEAED VGVYYC MQAWHSPT FGQGTKVEIKRT 6E10 VLRSSQSLLHSNGYNYLD  59 CDR1 6E10 VL LGSNRAS  60 CDR2 6E10 VL MQAWHSPT 242CDR3 12F9 VH QVQLVQSGAEVKKPGASVKVSCKASGYTF 130 aa T SYYMH WVRQAPGQGLEWMGIINPSGGST SYAQKFQG RVTMTRDTSTSTVYMELSSLR SEDTAVYYCARGATYTM DY WGQGTLVTVSSAS 12F9_VH_ SYYMH   8 CDR1 12F9_VH_ IINPSGGSTSYAQKFQG   9 CDR2 12F9_VH_GATYTMDY 243 CDR3 12F9 VL DIVMTQSPLSLPVTPGEPASISC RSSQSL 131 aaLHSNGYNYLD WYLQKPGQSPQLLIY LGSN RAS GVPDRFSGSGSGTDFTLKISRVEAED VGVYYCMQALQTPIT FGQGTKVEIKRT 12F9_VL_ RSSQSLLHSNGYNYLD  59 CDR1 12F9_VL_LGSNRAS  60 CDR2 12F9_VL_ MQALQTPIT 244 CDR3

10) 9D11 Glyscosite Variants: Variable Light Chain of ExemplaryEmbodiments (CDRs Underlined)

Variant Sequence Seq ID No N95S DIVMTQSPLSLPVTPGEPASISCRSSQS 132LLHSNGYNYLDWYLQKPGQSPQLLIYLG SNRASGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCMQASIMSRTFGQGTKVEIK 12F9_VL_ RSSQSLLHSNGYNYLD  59 CDR1 12F9_VL_LGSNRAS  60 CDR2 12F9_VL_ MQASIMSRT  63 CDR3 N95QDIVMTQSPLSLPVTPGEPASISCRSSQS 133 LLHSNGYNYLDWYLQKPGQSPQLLIYLGSNRASGVPDRFSGSGSGTDFTLKISRVE AEDVGVYYCMQASIMQRTFGQGTKVEIK N95Q_VL_RSSQSLLHSNGYNYLD  59 CDR1 N95Q_VL_ LGSNRAS  60 CDR2 N95Q_VL_ MQASIMQRT 65 CDR3 T97A DIVMTQSPLSLPVTPGEPASISCRSSQS 134LLHSNGYNYLDWYLQKPGQSPQLLIYLG SNRASGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCMQASIMNRA FGQGTKVEIK T97A_VL_ RSSQSLLHSNGYNYLD  59 CDR1T97A_VL_ LGSNRAS  60 CDR2 T97A_VL_ MQASIMNRA  67 CDR3 T97NDIVMTQSPLSLPVTPGEPASISCRSSQS 135 LLHSNGYNYLDWYLQKPGQSPQLLIYLGSNRASGVPDRFSGSGSGTDFTLKISRVE AEDVGVYYCMQASIMNRN FGQGTKVEIK T97N_VL_RSSQSLLHSNGYNYLD  59 CDR1 T97N_VL_ LGSNRAS  60 CDR2 T97N_VL_ MQASIMNRN 69 CDR3

11) Deamination Variants

Variant Sequence Seq ID No 16D5EVQLVESGGGLVKPGGSLRLSCAASGFTFSNAWMSWVRQAPGKGLEWVGRIK 248 VH_D52dESKTEGGTTDYAAPVKGRFTISRDDSKNTLYLQMNSLKTEDTAVYYCTTPWEW SWYDYWGQGTLVTVSS16D5 EVQLVESGGGLVKPGGSLRLSCAASGFTFSNAWMSWVRQAPGKGLEWVGRIK 249 VH_D52dQSKTQGGTTDYAAPVKGRFTISRDDSKNTLYLQMNSLKTEDTAVYYCTTPWEW SWYDYWGQGTLVTVSSCD3_VH EVQLLESGGGLVQPGGSLRLSCAASGFTFSTYAMNWVRQAPGKGLEWVSRIR 250 N100ASKYNNYATYYADSVKGRFTISRDDSKNTLYLQMNSLRAEDTAVYYCVRHGNFGASYVSWFAYWGQGTLVTVSS CD3_VHEVQLLESGGGLVQPGGSLRLSCAASGFTFSTYAMNWVRQAPGKGLEWVSRIR 251 S100aASKYNNYATYYADSVKGRFTISRDDSKNTLYLQMNSLRAEDTAVYYCVRHGNFGNAYVSWFAYWGQGTLVTVSS 16D5EVQLVESGGGLVKPGGSLRLSCAASGFTFSNAWMSWVRQAPGKGLEWVGRIK 252 [VHCH1]-SKTDGGTTDYAAPVKGRFTISRDDSKNTLYLQMNSLKTEDTAVYYCTTPWEW CD3SWYDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEP [VHCH1-VTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHK N100A]-PSNTKVDKKVEPKSCDGGGGSGGGGSEVQLLESGGGLVQPGGSLRLSCAASG Fcknob_FTFSTYAMNWVRQAPGKGLEWVSRIRSKYNNYATYYADSVKGRFTISRDDSK PGLALANTLYLQMNSLRAEDTAVYYCVRHGNFGASYVSWFAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHN HYTQKSLSLSPGK 16D5-EVQLVESGGGLVKPGGSLRLSCAASGFTFSNAWMSWVRQAPGKGLEWVGRIK 253 Fchole-SKTDGGTTDYAAPVKGRFTISRDDSKNTLYLQMNSLKTEDTAVYYCTTPWEW PGLALASWYDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNRFTQKSLSLSPGK CD3-CLCQAVVTQEPSLTVSPGGTVTLTCGSSTGAVTTSNYANWVQEKPGQAFRGLIGG 254TNKRAPGTPARESGSLLGGKAALTLSGAQPEDEAEYYCALWYSNLWVFGGGTKLTVLGQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKT VAPTECS 16D5EVQLVESGGGLVKPGGSLRLSCAASGFTFSNAWMSWVRQAPGKGLEWVGRIK 255 [VHCH1]-SKTDGGTTDYAAPVKGRFTISRDDSKNTLYLQMNSLKTEDTAVYYCTTPWEW CD3SWYDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEP [VHCH1-VTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHK S100aA]-PSNTKVDKKVEPKSCDGGGGSGG Fcknob_GGSEVQLLESGGGLVQPGGSLRLSCAASGETFSTYAMNWVRQAPGKGLEWVS PGLALARIRSKYNNYATYYADSVKGRFTISRDDSKNTLYLQMNSLRAEDTAVYYCVRHGNFGNAYVSWFAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 9D11QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYYMHWVRQAPGQGLEWMGIIN 256 [VHCH1]-PSGGPTSYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARGDFAWL CD3DYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTV [VHCL-SWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSN N100A]-TKVDKKVEPKSCDGGGGSGGGGSEVQLLESGGGLVQPGGSLRLSCAASGFTF Fcknob_STYAMNWVRQAPGKGLEWVSRIRSKYNNYATYYADSVKGRFTISRDDSKNTL PGLALAYLQMNSLRAEDTAVYYCVRHGNFGASYVSWFAYWGQGTLVTVSSASVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGECDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALH NHYTQKSLSLSPGK9D11- QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYYMHWVRQAPGQGLEWMGIIN 257 FcholePSGGPTSYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARGDFAWLDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 9D11_LCDIVMTQSPLSLPVTPGEPASISCRSSQSLLHSNGYNYLDWYLQKPGQSPQLL 258 [N95Q]IYLGSNRASGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCMQASIMQRTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSS PVTKSFNRGEC CD3_QAVVTQEPSLTVSPGGTVTLTCGSSTGAVTTSNYANWVQEKPGQAFRGLIGG 259 VLCH1TNKRAPGTPARESGSLLGGKAALTLSGAQPEDEAEYYCALWYSNLWVFGGGTKLTVLSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKK VEPKSC 9D11QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYYMHWVRQAPGQGLEWMGIIN 260 [VHCH1]-PSGGPTSYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARGDFAWL CD3DYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTV [VHCH1-SWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSN S100aA]-TKVDKKVEPKSCDGGGGSGGGGSEVQLLESGGGLVQPGGSLRLSCAASGFTF Fcknob_STYAMNWVRQAPGKGLEWVSRIRSKYNNYATYYADSVKGRFTISRDDSKNTL PGLALAYLQMNSLRAEDTAVYYCVRHGNFGNAYVSWFAYWGQGTLVTVSSASVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGECDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALH NHYTQKSLSLSPGK

12) Mov19 Based TCBs of Exemplary Embodiments (CDRs Underlined)

Name Sequence Seq ID No pETR11646 QVQLQQSGAELVKPGASVKISCKASGYSFT GYFMNWVKQSHGKSLEWIG RIH 136 Mov19 PYDGDTFYNQNFKDKATLTVDKSSNTAHMELLSLTSEDFAVYYCTR YDGSRA VH-CH1- MDYWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVT FcholeVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPS PG/LALANTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK pETR11647QVQLQQSGAELVKPGASVKISCKASGYSFT GYFMN WVKQSHGKSLEWIG RIH 137 Mov19PYDGDTFYNQNFKD KATLTVDKSSNTAHMELLSLTSEDFAVYYCTR YDGSRA VH-CH1- MDYWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVT CD3 VH-VSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPS CL-NTKVDKKVEPKSCDGGGGSGGGGSEVQLVESGGGLVQPKGSLKLSCAAS GFT Fcknob FNTYAMNWVRQAPGKGLEWVA RIRSKYNNYATYYADSVKD RFTISRDDSQSI PG/LALALYLQMNNLKTEDTAMYYCVR HGNFGNSYVSWFAY WGQGTLVTVSAASVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGECDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEAL HNHYTQKSLSLSPGKpETR11644 DIELTQSPASLAVSLGQRAIISC KASQSVSFAGTSLMH WYHQKPGQQPKLLI 138Mov19 LC Y RASNLEA GVPTRFSGSGSKTDFTLNIHPVEEEDAATYYC QQSREYPYT FGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSP VTKSFNRGEC Hu IgG1DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPE 245 FcVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVM HEALHNHYTQKSLSLSPGK

13) Additional FolR1 TCBs with Intermediate Affinity Binders (CDRsAccording to Kabat, Underlined):

Name Sequence Seq ID No 16D5 variantEVQLVESGGGLVKPGGSLRLSCAASGFTFSNAWMSWVRQAPGKGLEWVGRIKSK 274 W96Y/D52ETEGGTTDYAAPVKGRFTISRDDSKNTLYLQMNSLKTEDTAVYYCTTPYEWSWYD VH YWGQGTLVTVSSW96Y/D52E_ NAWMS  16 VH CDR1 W96Y/D52E_ RIKSKTEGGTTDYAAPVKG 275 VH CD R2W96Y/D52E_ PYEWSWYDY 315 VH CD R3 16D5 variantQAVVTQEPSLTVSPGGTVTLTCGSSTGAVTTSNYANWVQEKPGQA  31 W96Y/D52EFRGLIGGTNKRAPGTPARFSGSLLGGKAALTLSGAQPEDEAEYYC VL ALWYSNLWVFGGGTKLTVLW96Y/D52E_ EVQLVESGGGLVKPGGSLRLSCAASGFTFSNAWMSWVRQAPGKGLEWVGRIKSK 276CD3- TEGGTTDYAAPVKGRFTISRDDSKNTLYLQMNSLKTEDTAVYYCTTPYEWSWYD VHCH1_Fc_YWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWN knob_PGLALASGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDK pETR14945KVEPKSCDGGGGSGGGGSEVQLLESGGGLVQPGGSLRLSCAASGFTFSTYAMNWVRQAPGKGLEWVSRIRSKYNNYATYYADSVKGRFTISRDDSKNTLYLQMNSLRAEDTAVYYCVRHGNFGNSYVSWFAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK W96Y/D52E_EVQLVESGGGLVKPGGSLRLSCAASGETFSNAWMSWVRQAPGKGLEWVGRIKSK 277 Fc-hole_TEGGTTDYAAPVKGRFTISRDDSKNTLYLQMNSLKTEDTAVYYCTTPYEWSWYD PGLALA_YWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWN HYRFSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDK pETR14946KVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFELVSKLTVDKSRWQQGNVESCSVMHEALHNRFTQKSLSLSPGK 14B1 EVQLLESGGGLVQPGGSLRLSCAASGFTFS SYAMSWVRQAPGKGLEWVS AISGS 278 VH GGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAR GDYRYRYFDY WGQGTLVTVSS 14B1SSELTQDPAVSVALGQTVRITC QGDSLRSYYAS WYQQKPGQAPVLVIY GKNNRP 279 VL SGIPDRFSGSSSGNTASLTITGAQAEDEADYYC NSRESPPTGLVV FGGGTKLTV L 14B1[EE]_EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAISGS 280 CD3[VLCH1]_GGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARGDYRYRYFDY Fc-knob_WGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVEDYFPEPVTVSWNS PGLALAGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDEK pETR14976VEPKSCDGGGGSGGGGSQAVVTQEPSLTVSPGGTVTLTCGSSTGAVTTSNYANWVQEKPGQAFRGLIGGTNKRAPGTPARESGSLLGGKAALTLSGAQPEDEAEYYCALWYSNLWVFGGGTKLTVLSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 14B1[EE]_EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAISGS 281 Fc-hole_GGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARGDYRYRYFDY PGLALAWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVEDYFPEPVTVSWNS pETR14977GALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDEKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFELVSKLTVDKSRWQQGNVESCSVMHE ALHNHYTQKSLSLSPGK14B1 LC SSELTQDPAVSVALGQTVRITCQGDSLRSYYASWYQQKPGQAPVLVIYGKNNRP 282 [KK]SGIPDRFSGSSSGNTASLTITGAQAEDEADYYCNSRESPPTGLVVFGGGTKLTV ConstantLGQPKAAPSVTLFPPSSKKLQANKATLVCLISDFYPGAVTVAWKADSSPVKAGV lambdaETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS pETR14979 9C7 VHQVQLVQSGAEVKKPGASVKVSCKASGYTFT SYYMH WVRQAPGQGLEWMGI INPS 283GGSTSYAQKFQG RVTMTRDTSTSTVYMELSSLRSEDTAVYYCAR GDWSYYMDY W GQGTLVTVSS9C7 VL DIVMTQSPLSLPVTPGEPASISC RSSQSLLHSNGYNYLD WYLQKPGQSPQLLIY 284LGSNRAS GVPDRFSGSGSGTDFTLKISRVEAEDVGVYYC MQARQTPT FGQGTKV EIK 9C7[EE]_QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYYMHWVRQAPGQGLEWMGIINPS 285 CD3[VLCH1]_GGSTSYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARGDWSYYMDYW Fe-knob_GQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVEDYFPEPVTVSWNSG PGLALAALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDEKV pETR14974EPKSCDGGGGSGGGGSQAVVTQEPSLTVSPGGTVTLTCGSSTGAVTTSNYANWVQEKPGQAFRGLIGGTNKRAPGTPARESGSLLGGKAALTLSGAQPEDEAEYYCALWYSNLWVFGGGTKLTVLSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 9C7[EE]_QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYYMHWVRQAPGQGLEWMGIINPS 286 Fc_hole_GGSTSYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARGDWSYYMDYW PGLALAGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVEDYFPEPVTVSWNSG pETR14975ALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDEKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFELVSKLTVDKSRWQQGNVESCSVMHEA LHNHYTQKSLSLSPGK9C7 LC DIVMTQSPLSLPVTPGEPASISCRSSQSLLHSNGYNYLDWYLQKPGQSPQLLIY 316 [RK]LGSNRASGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCMQARQTPTFGQGTKV pETR14980EIKRTVAAPSVFIFPPSDRKLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRG EC

14) Antigen Sequences

Antigen Sequence Seq ID No hu FolR1MAQRMTTQLLLLLVWVAVVGEAQTRIAWARTELLNVCMNAKHHKEKPGPEDKL 139HEQCRPWRKNACCSTNTSQEAHKDVSYLYRFNWNHCGEMAPACKRHFIQDTCLYECSPNLGPWIQQVDQSWRKERVLNVPLCKEDCEQWWEDCRTSYTCKSNWHKGWNWTSGENKCAVGAACQPFHFYFPTPTVLCNEIWTHSYKVSNYSRGSGRCIQMWFDPAQGNPNEEVARFYAAAMSGAGPWAAWPFLLSLALMLLWLLS huFolR1RIAWARTELLNVCMNAKHHKEKPGPEDKLHEQCRPWRKNACCSTNTSQEAHKD 140 ECD-VSYLYRFNWNHCGEMAPACKRHFIQDTCLYECSPNLGPWIQQVDQSWRKERVL AcTev-NVPLCKEDCEQWWEDCRTSYTCKSNWHKGWNWTSGENKCAVGAACQPFHFYFP Fcknob-TPTVLCNEIWTHSYKVSNYSRGSGRCIQMWFDPAQGNPNEEVARFYAAAMVD E Avi tag QLYFQGGSPKSADKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTC VVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKSGGLNDIFEAQKIEWHE FcholeDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEV 141KFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFELVSKLTVDKSRWQQGNVESCSVMHEAL HNRFTQKSLSLSPGK MUMAHLMTVQLLLLVMWMAECAQSRATRARTELLNVCMDAKHHKEKPGPEDNLHD 142 FolR1QCSPWKTNSCCSTNTSQEAHKDISYLYRFNWNHCGTMTSECKRHFIQDTCLYECSPNLGPWIQQVDQSWRKERILDVPLCKEDCQQWWEDCQSSFTCKSNWHKGWNWSSGHNECPVGASCHPFTFYFPTSAALCEEIWSHSYKLSNYSRGSGRCIQMWFDPAQGNPNEEVARFYAEAMSGAGLHGTWPLLCSLSLVLLWVIS MUTRARTELLNVCMDAKHHKEKPGPEDNLHDQCSPWKTNSCCSTNTSQEAHKDIS 143 FolR1YLYRFNWNHCGTMTSECKRHFIQDTCLYECSPNLGPWIQQVDQSWRKERILDV ECD-PLCKEDCQQWWEDCQSSFTCKSNWHKGWNWSSGHNECPVGASCHPFTFYFPTS AcTev-AALCEEIWSHSYKLSNYSRGSGRCIQMWFDPAQGNPNEEVARFYAEAMVD EQL Fcknob- YFQGGSPKSADKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVV AvitagVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKSGGLNDIFEAQKIEWHE cy FolR1MAQRMTTQLLLLLVWVAVVGEAQTRTARARTELLNVCMNAKHHKEKPGPEDKL 144HEQCRPWKKNACCSTNTSQEAHKDVSYLYRENWNHCGEMAPACKRHFIQDTCLYECSPNLGPWIQQVDQSWRKERVLNVPLCKEDCERWWEDCRTSYTCKSNWHKGWNWTSGENKCPVGAACQPFHEYEPTPTVLCNEIWTYSYKVSNYSRGSGRCIQMWFDPAQGNPNEEVARFYAAAMSGAGPWAAWPLLLSLALTLLWLLS cy FolR1RTARARTELLNVCMNAKHHKEKPGPEDKLHEQCRPWKKNACCSTNTSQEAHKD 145 ECD-VSYLYRFNWNHCGEMAPACKRHFIQDTCLYECSPNLGPWIQQVDQSWRKERVL AcTev-NVPLCKEDCEQWWEDCRTSYTCKSNWHKGWNWTSGENKCPVGAACQPFHFYFP Fcknob-TPTVLCNEIWTYSYKVSNYSRGSGRCIQMWFDPAQGNPNEEVARFYAAAMVD E Avi tag QLYFQGGSPKSADKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTC VVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKSGGLNDIFEAQKIEWHE hu FolR2MVWKWMPLLLLLVCVATMCSAQDRTDLLNVCMDAKHHKTKPGPEDKLHDQCSP 146WKKNACCTASTSQELHKDTSRLYNENWDHCGKMEPACKRHFIQDTCLYECSPNLGPWIQQVNQSWRKERFLDVPLCKEDCQRWWEDCHTSHTCKSNWHRGWDWTSGVNKCPAGALCRTFESYFPTPAALCEGLWSHSYKVSNYSRGSGRCIQMWFDSAQGNPNEEVARFYAAAMHVNAGEMLHGTGGLLLSLALMLQLWLLG hu FolR2TMCSAQDRTDLLNVCMDAKHHKTKPGPEDKLHDQCSPWKKNACCTASTSQELH 147 ECD-KDTSRLYNFNWDHCGKMEPACKRHFIQDTCLYECSPNLGPWIQQVNQSWRKER AcTev-FLDVPLCKEDCQRWWEDCHTSHTCKSNWHRGWDWTSGVNKCPAGALCRTFESY Fcknob-FPTPAALCEGLWSHSYKVSNYSRGSGRCIQMWFDSAQGNPNEEVARFYAAAMH Avi tag VVDEQLYFQG GSPKSADKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKSGGLNDIFEAQKIEWHE hu FolR3MAWQMMQLLLLALVTAAGSAQPRSARARTDLLNVCMNAKHHKTQPSPEDELYG 148QCSPWKKNACCTASTSQELHKDTSRLYNFNWDHCGKMEPTCKRHFIQDSCLYECSPNLGPWIRQVNQSWRKERILNVPLCKEDCERWWEDCRTSYTCKSNWHKGWNWTSGINECPAGALCSTFESYFPTPAALCEGLWSHSEKVSNYSRGSGRCIQMWFDSAQGNPNEEVAKFYAAAMNAGAPSRGIIDS hu FolR3SARARTDLLNVCMNAKHHKTQPSPEDELYGQCSPWKKNACCTASTSQELHKDT 149 ECD-SRLYNFNWDHCGKMEPTCKRHFIQDSCLYECSPNLGPWIRQVNQSWRKERILN AcTev-VPLCKEDCERWWEDCRTSYTCKSNWHKGWNWTSGINECPAGALCSTFESYFPT Fcknob-PAALCEGLWSHSFKVSNYSRGSGRCIQMWFDSAQGNPNEEVAKFYAAAMNAGA Avi tag PSRGIIDSVDEQLYFQG GSPKSADKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKSGGLNDIFEAQKIEW HE hu CD3εMQSGTHWRVLGLCLLSVGVWGQDGNEEMGGITQTPYKVSISGTTVILTCPQYP 150GSEILWQHNDKNIGGDEDDKNIGSDEDHLSLKEFSELEQSGYYVCYPRGSKPEDANFYLYLRARVCENCMEMDVMSVATIVIVDICITGGLLLLVYYWSKNRKAKAKPVTRGAGAGGRQRGQNKERPPPVPNPDYEPIRKGQRDLYSGLNQRRI

15) Nucleotide Sequences of Exemplary Embodiments

Seq Description Sequence ID No 16A3CAGGTGCAATTGGTTCAATCTGGTGCTGAAGTAAAAAAACCGGGCG 151CTTCCGTTAAAGTGAGCTGCAAAGCATCCGGATACACCTTCACTTCCTATTACATGCACTGGGTTCGTCAAGCCCCGGGCCAGGGTCTGGAATGGATGGGCATCATTAACCCAAGCGGTGGCTCTACCTCCTACGCGCAGAAATTCCAGGGTCGCGTCACGATGACCCGTGACACTAGCACCTCTACCGTTTATATGGAGCTGTCCAGCCTGCGTTCTGAAGATACTGCAGTGTACTACTGTGCACGCAACTACTACGCTGGTGTTACTCCGTTCGACTATTGGGGTCAAGGCACCCTCGTAACGGTTTCTTCT 15A1CAGGTGCAATTGGTTCAATCTGGTGCTGAAGTAAAAAAACCGGGCG 152CTTCCGTTAAAGTGAGCTGCAAAGCATCCGGATACACCTTCACTTCCTATTACATGCACTGGGTTCGTCAAGCCCCGGGCCAGGGTCTGGAATGGATGGGCATCATTAACCCAAGCGGTGGCTCTACCTCCTACGCGCAGAAATTCCAGGGTCGCGTCACGATGACCCGTGACACTAGCACCTCTACCGTTTATATGGAGCTGTCCAGCCTGCGTTCTGAAGATACTGCAGTGTACTACTGTGCACGCAACTACTACATCGGTGTTGTTACTTTCGACTATTGGGGTCAAGGCACCCTCGTAACGGTTTCTTCT 18D3CAGGTGCAATTGGTTCAATCTGGTGCTGAAGTAAAAAAACCGGGCG 153CTTCCGTTAAAGTGAGCTGCAAAGCATCCGGATACACCTTCACTTCCTATTACATGCACTGGGTTCGTCAAGCCCCGGGCCAGGGTCTGGAATGGATGGGCATCATTAACCCAAGCGGTGGCTCTACCTCCTACGCGCAGAAATTCCAGGGTCGCGTCACGATGACCCGTGACACTAGCACCTCTACCGTTTATATGGAGCTGTCCAGCCTGCGTTCTGAAGATACTGCAGTGTACTACTGTGCACGCAACTACTACACTGGTGGTTCTTCTGCTTTCGACTATTGGGGTCAAGGCACCCTCGTAACGGTTTCTTCT 19E5CAGGTGCAATTGGTTCAATCTGGTGCTGAAGTAAAAAAACCGGGCG 154NTTCCGTTAAAGTGAGCTGCAAAGCATCCGGATACACCTTCACTTCCTATTACATGCACTGGGTTCGTCAAGCCCCGGGCCAGGGTCTGGAATGGATGGGCATCATTAACCCAAGCGGTGGCTCTACCTCCTACGCGCAGAAATTCCAGGGTCGCGTCACGATGACCCGTGACACTAGCACCTCTACCGTTTATATGGAGCTGTCCAGCCTGCGTTCTGAAGATACTGCAGTGTACTACTGTGCACGCGGTGAATGGCGTCGTTACACTTCTTTCGACTATTGGGGTCAAGGCACCCTCGTAACGGTTTCTTCT 19A4CAGGTGCAATTGGTTCAATCTGGTGCTGAAGTAAAAAAACCGGGCG 155CTTCCGTTAAAGTGAGCTGCAAAGCATCCGGATACACCTTCACTTCCTATTACATGCACTGGGTTCGTCAAGCCCCGGGCCAGGGTCTGGAATGGATGGGCATCATTAACCCAAGCGGTGGCTCTACCTCCTACGCGCAGAAATTCCAGGGTCGCGTCACGATGACCCGTGACACTAGCACCTCTACCGTTTATATGGAGCTGTCCAGCCTGCGTTCTGAAGATACTGCAGTGTACTACTGTGCACGCGGTGGTTGGATCCGTTGGGAACATTTCGACTATTGGGGTCAAGGCACCCTCGTAACGGTTTCTTCT 15H7CAGGTGCAATTGGTTCAATCTGGTGCTGAAGTAAAAAAACCGGGCG 156CTTCCGTTAAAGTGAGCTGCAAAGCATCCGGATACACCTTCACTTCCTATTACATGCACTGGGTTCGTCAAGCCCCGGGCCAGGGTCTGGAATGGATGGGCATCATTAACCCAAGCGGTGGCTCTACCTCCTACGCGCAGAAATTCCAGGGTCGCGTCACGATGACCCGTGACACTAGCACCTCTACCGTTTATATGGAGCTGTCCAGCCTGCGTTCTGAAGATACTGCAGTGTACTACTGTGCACGCAACTACTACCTGTTCTCTACTTCTTTCGACTATTGGGGTCAAGGCACCCTCGTAACGGTTTCTTCT 15B6CAGGTGCAATTGGTTCAATCTGGTGCTGAGGTAAAAAAACCGGGCG 157CTTCCGTTAAAGTGAGCTGCAAAGCATCCGGATACACCTTCACTTCCTATTACATGCACTGGGTTCGTCAAGCCCCGGGCCAGGGTCTGGAATGGATGGGCATCATTAACCCAAGCGGTGGCTCTACCTCCTACGCGCAGAAATTCCAGGGTCGCGTCACGATGACCCGTGACACTAGCACCTCTACCGTTTATATGGAGCTGTCCAGCCTGCGTTCTGAAGATACTGCAGTGTACTACTGTGCACGCAACTACTACATCGGTATCGTTCCGTTCGACTATTGGGGTCAAGGCACCCTCGTAACGGTTTCTTCT 16D5GAGGTGCAATTGGTTGAATCTGGTGGTGGTCTGGTAAAACCGGGCG 158GTTCCCTGCGTCTGAGCTGCGCGGCTTCCGGATTCACCTTCTCCAACGCGTGGATGAGCTGGGTTCGCCAGGCCCCGGGCAAAGGCCTCGAGTGGGTTGGTCGTATCAAGTCTAAAACTGACGGTGGCACCACGGATTACGCGGCTCCAGTTAAAGGTCGTTTTACCATTTCCCGCGACGATAGCAAAAACACTCTGTATCTGCAGATGAACTCTCTGAAAACTGAAGACACCGCAGTCTACTACTGTACTACCCCGTGGGAATGGTCTTGGTACGATTATTGGGGCCAGGGCACGCTGGTTACGGTGTCTTCC 15E12GAGGTGCAATTGGTTGAATCTGGTGGTGGTCTGGTAAAACCGGGCG 159GTTCCCNGCGTCTGAGCTGCGCGGCTTCCGGATTCACCTTCTCCAACGCGTGGATGAGCTGGGTTCGCCAGGCCCCGGGCAAAGGCCTCGAGTGGGTTGGTCGTATCAAGTCTAAAACTGACGGTGGCACCACGGATTACGCGGCTCCAGTTAAAGGTCGTTTTACCATTTCCCGCGACGATAGCAAAAACACTCTGTATCTGCAGATGAACTCTCTGAAAACCGAAGACACCGCAGTCTACTACTGTACTACCCCGTGGGAATGGTCTTACTTCGATTATTGGGGCCAGGGCACGCTGGTTACGGTGTCTTCC 21D1CAGGTGCAATTGGTTCAATCTGGTGCTGAAGTAAAAAAACCGGGCG 160CTTCCGTTAAAGTGAGCTGCAAAGCATCCGGATACACCTTCACTTCCTATTACATGCACTGGGTTCGTCAAGCCCCGGGCCAGGGTCTGGAATGGATGGGCATCATTAACCCAAGCGGTGGCTCTACCTCCTACGCGCAGAAATTCCAGGGTCGCGTCACGATGACCCGTGACACTAGCACCTCTACCGTTTATATGGAGCTGTCCAGCCTGCGTTCTGAAGATACTGCAGTGTACTACTGTGCACGCAACTACTACGTTGGTGTTTCTCCGTTCGACTATTGGGGTCAAGGCACCCTCGTAACGGTTTCTTCT 16F12CAGGTGCAATTGGTTCAATCTGGTGCTGAAGTAAAAAAACCGGGCG 161NTTCCGTTAAAGTGAGCTGCAAAGCATCCGGATACACCTTCACTTCCTATTACATGCACTGGGTTCGTCAAGCCCCGGGCCAGGGTCTGGAATGGATGGGCATCATTAACCCAAGCGGTGGCTCTACCTCNTACGCGCAGAAATTCCAGGGTCGCGTCACGATGACCCGTGACACTAGCACCTCTACCGTTTATATGGAGCTGTCCAGCCTGCGTTCTGAAGATACTGCAGTGTACTACTGTGCACGCAACTTCACTGTTCTGCGTGTTCCGTTCGACTATTGGGGTCAAGGCACCCTCGTAACGGTTTCTTCT 21A5GAGGTGCAATTGGTTGAATCTGGTGGTGGTCTGGTAAAACCGGGCG 162GTTCCCTGCGTCTGAGCTGCGCGGCTTCCGGATTCACCTTCTCCAACGCGTGGATGAGCTGGGTTCGCCAGGCCCCGGGCAAAGGCCTCGAGTGGGTTGGTCGTATCAAGTCTAAAACTGACGGTGGCACCACGGATTACGCGGCTCCAGTTAAAGGTCGTTTTACCATTTCCCGCGACGATAGCAAAAACACTCTGTATCTGCAGATGAACTCTCTGAAAACTGAAGACACCGCAGTCTACTACTGTACTACCCCGTGGGAATGGGCTTGGTTCGATTATTGGGGCCAGGGCACGCTGGTTACGGTGTCTTCC 21G8GAGGTGCAATTGGTTGAATCTGGTGGTGGTCTGGTAAAACCGGGCG 163GTTCCCTGCGTCTGAGCTGCGCGGCTTCCGGATTCACCTTCTCCAACGCGTGGATGAGCTGGGTTCGCCAGGCCCCGGGCAAAGGCCTCGAGTGGGTTGGTCGTATCAAGTCTAAAACTGACGGTGGCACCACGGATTACGCGGCTCCAGTTAAAGGTCGTTTTACCATTTCCCGCGACGATAGCAAAAACACTCTGTATCTGCAGATGAACTCTCTGAAAACCGAAGACACCGCAGTCTACTACTGTACTACCCCTTGGGAATGGGCTTACTTCGATTATTGGGGCCAGGGCACGCTGGTTACGGTGTCTTCC 19H3CAGGTGCAATTGGTTCAATCTGGTGCTGAAGTAAAAAAACCGGGCG 164CTTCCGTTAAAGTGAGCTGCAAAGCATCCGGATACACCTTCACTTCCTATTACATGCACTGGGTTCGTCAAGCCCCGGGCCAGGGTCTGGAATGGATGGGCATCATTAACCCAAGCGGTGGCTCTACCTCCTACGCGCAGAAATTCCAGGGTCGCGTCACGATGACCCGTGACACTAGCACCTCTACCGTTTATATGGAGCTGTCCAGCCTGCGTTCTGAAGATACTGCAGTGTACTACTGTGCACGCACTGGTTGGTCTCGTTGGGGTTACATGGACTATTGGGGCCAAGGCACCCTCGTAACGGTTTCTTCT 20G6CAGGTGCAATTGGTTCAATCTGGTGCTGAAGTAAAAAAACCGGGCG 165CTTCCGTTAAAGTGAGCTGCAAAGCATCCGGATACACCTTCACTTCCTATTACATGCACTGGGTTCGTCAAGCCCCGGGCCAGGGTCTGGAATGGATGGGCATCATTAACCCAAGCGGTGGCTCTACCTCCTACGCGCAGAAATTCCAGGGTCGCGTCACGATGACCCGTGACACTAGCACCTCTACCGTTTATATGGAGCTGTCCAGCCTGCGTTCTGAAGATACTGCAGTGTACTACTGTGCACGCGGTGAATGGATCCGTTACTACCATTTCGACTATTGGGGTCAAGGCACCCTCGTAACGGTTTCTTCT 20H7CAGGTGCAATTGGTTCAATCTGGTGCTGAAGTAAAAAAACCGGGCG 166CTTCCGTTAAAGTGAGCTGCAAAGCATCCGGATACACCTTCACTTCCTATTACATGCACTGGGTTCGTCAAGCCCCGGGCCAGGGTCTGGAATGGATGGGCATCATTAACCCAAGCGGTGGCTCTACCTCCTACGCGCAGAAATTCCAGGGTCGCGTCACGATGACCCGTGACACTAGCACCTCTACCGTTTATATGGAGCTGTCCAGCCTGCGTTCTGAAGATACTGCAGTGTACTACTGTGCACGCGTTGGTTGGTACCGTTGGGGTTACATGGACTATTGGGGTCAAGGCACCCTCGTAACGGTTTCTTCT 11F8_VHCAGGTGCAATTGGTGCAGTCTGGGGCTGAGGTGAAGAAGCCTGGGT 167CCTCGGTGAAGGTCTCCTGCAAGGCCTCCGGAGGCACATTCAGCAGCTACGCTATAAGCTGGGTGCGACAGGCCCCTGGACAAGGGCTCGAGTGGATGGGAGGGATCATCCCTATCTTTGGTACAGCAAACTACGCACAGAAGTTCCAGGGCAGGGTAACCATTACTGCAGACAAATCCACGAGCACAGCCTACATGGAGCTGAGCAGCCTGAGATCTGAGGACACCGCCGTGTATTACTGTGCGAGAGCTGTTTTCTACCGTGCTTGGTACTCTTTCGACTACTGGGGCCAAGGGACCACCGTGACCGTCTCCTCA 11F8_VLGACATCCAGATGACCCAGTCTCCTTCCACCCTGTCTGCATCTGTAG 168GAGACCGTGTCACCATCACTTGCCGTGCCAGTCAGAGTATTAGTAGCTGGTTGGCCTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTATGATGCCTCCAGTTTGGAAAGTGGGGTCCCATCACGTTTCAGCGGCAGTGGATCCGGGACAGAATTCACTCTCACCATCAGCAGCTTGCAGCCTGATGATTTTGCAACTTATTACTGCCAACAGTATACCAGCCCACCACCAACGTTTGGCCAGGGCACCAAAGTCGAGATCAAG 36F2_VHCAGGTGCAATTGGTTCAATCTGGTGCTGAAGTAAAAAAACCGGGCG 169CTTCCGTTAAAGTGAGCTGCAAAGCATCCGGATACACCTTCACTTCCTATTACATGCACTGGGTTCGTCAAGCCCCGGGCCAGGGTCTGGAATGGATGGGCATCATTAACCCAAGCGGTGGCTCTACCTCCTACGCGCAGAAATTCCAGGGTCGCGTCACGATGACCCATGACACTAGCACCTCTACCGTTTATATGGAGCTGTCCAGCCTGCGTTCTGAAGATACTGCAGTGTACTACTGTGCACGCTCTTTCTTCACTGGTTTCCATCTGGACTATTGGGGTCAAGGCACCCTCGTAACGGTTTCTTCT 36F2_VLGAAATCGTGTTAACGCAGTCTCCAGGCACCCTGTCTTTGTCTCCAG 170GGGAAAGAGCCACCCTCTCTTGCAGGGCCAGTCAGAGTGTTAGCAGCAGCTACTTAGCCTGGTACCAGCAGAAACCTGGCCAGGCTCCCAGGCTCCTCATCTATGGAGCATCCAGCAGGGCCACTGGCATCCCAGACAGGTTCAGTGGCAGTGGATCCGGGACAGACTTCACTCTCACCATCAGCAGACTGGAGCCTGAAGATTTTGCAGTGTATTACTGTCAGCAGTATACCAACGAACATTATTATACGTTCGGCCAGGGGACCAAAGTGGAAA TCAAA 9D11_VHCAGGTGCAATTGGTTCAATCTGGTGCTGAAGTAAAAAAACCGGGCG 171CTTCCGTTAAAGTGAGCTGCAAAGCATCCGGATACACCTTCACTTCCTATTACATGCACTGGGTTCGTCAAGCCCCGGGCCAGGGTCTGGAATGGATGGGCATCATTAACCCAAGCGGTGGCCCTACCTCCTACGCGCAGAAATTCCAGGGTCGCGTCACGATGACCCGTGACACTAGCACCTCTACCGTTTATATGGAGCTGTCCAGCCTGCGTTCTGAAGATACTGCAGTGTACTACTGTGCACGCGGTGACTTCGCTTGGCTGGACTATTGGGGTCAAGGCACCCTCGTAACGGTTTCTTCT 9D11_VLGATATTGTTATGACTCAATCTCCACTGTCTCTGCCGGTGACTCCAG 172GCGAACCGGCGAGCATTTCTTGCCGTTCCAGCCAGTCTCTGCTGCACTCCAACGGCTACAACTATCTCGATTGGTACCTGCAAAAACCGGGTCAGAGCCCTCAGCTGCTGATCTACCTGGGCTCTAACCGCGCTTCCGGTGTACCGGACCGTTTCAGCGGCTCTGGATCCGGCACCGATTTCACGTTGAAAATCAGCCGTGTTGAAGCAGAAGACGTGGGCGTTTATTACTGTATGCAGGCAAGCATTATGAACCGGACTTTTGGTCAAGGCACCA AGGTCGAAATTAAA 9D11_VLGATATTGTTATGACTCAATCTCCACTGTCTCTGCCGGTGACTCCAG 173 N95SGCGAACCGGCGAGCATTTCTTGCCGTTCCAGCCAGTCTCTGCTGCACTCCAACGGCTACAACTATCTCGATTGGTACCTGCAAAAACCGGGTCAGAGCCCTCAGCTGCTGATCTACCTGGGCTCTAACCGCGCTTCCGGTGTACCGGACCGTTTCAGCGGCTCTGGATCCGGCACCGATTTCACGTTGAAAATCAGCCGTGTTGAAGCAGAAGACGTGGGCGTTTATTACTGTATGCAGGCAAGCATTATGAGCCGGACTTTTGGTCAAGGCACCA AGGTCGAAATTAAA 9D11_VLGATATTGTTATGACTCAATCTCCACTGTCTCTGCCGGTGACTCCAG 174 N95QGCGAACCGGCGAGCATTTCTTGCCGTTCCAGCCAGTCTCTGCTGCACTCCAACGGCTACAACTATCTCGATTGGTACCTGCAAAAACCGGGTCAGAGCCCTCAGCTGCTGATCTACCTGGGCTCTAACCGCGCTTCCGGTGTACCGGACCGTTTCAGCGGCTCTGGATCCGGCACCGATTTCACGTTGAAAATCAGCCGTGTTGAAGCAGAAGACGTGGGCGTTTATTACTGTATGCAGGCAAGCATTATGCAGCGGACTTTTGGTCAAGGCACCA AGGTCGAAATTAAA 9D11_VLGATATTGTTATGACTCAATCTCCACTGTCTCTGCCGGTGACTCCAG 175 T97AGCGAACCGGCGAGCATTTCTTGCCGTTCCAGCCAGTCTCTGCTGCACTCCAACGGCTACAACTATCTCGATTGGTACCTGCAAAAACCGGGTCAGAGCCCTCAGCTGCTGATCTACCTGGGCTCTAACCGCGCTTCCGGTGTACCGGACCGTTTCAGCGGCTCTGGATCCGGCACCGATTTCACGTTGAAAATCAGCCGTGTTGAAGCAGAAGACGTGGGCGTTTATTACTGTATGCAGGCAAGCATTATGAACCGGGCTTTTGGTCAAGGCACCA AGGTCGAAATTAAA 9D11_VLGATATTGTTATGACTCAATCTCCACTGTCTCTGCCGGTGACTCCAG 176 T97NGCGAACCGGCGAGCATTTCTTGCCGTTCCAGCCAGTCTCTGCTGCACTCCAACGGCTACAACTATCTCGATTGGTACCTGCAAAAACCGGGTCAGAGCCCTCAGCTGCTGATCTACCTGGGCTCTAACCGCGCTTCCGGTGTACCGGACCGTTTCAGCGGCTCTGGATCCGGCACCGATTTCACGTTGAAAATCAGCCGTGTTGAAGCAGAAGACGTGGGCGTTTATTACTGTATGCAGGCAAGCATTATGAACCGGAATTTTGGTCAAGGCACCA AGGTCGAAATTAAA 5D9_VHCAGGTGCAATTGGTTCAATCTGGTGCTGAAGTAAAAAAACCGGGCG 177CTTCCGTTAAAGTGAGCTGCAAAGCATCCGGATACACCTTCACTTCCTATTACATGCACTGGGTTCGTCAAGCCCCGGGCCAGGGTCTGGAATGGATGGGCATCATTAACCCAAGCGGTGGCTCTACCTCCTACGCGCAGAAATTCCAGGGTCGCGTCACGATGACCCGTGACACTAGCACCTCTACCGTTTATATGGAGCTGTCCAGCCTGCGTTCTGAAGATACTGCAGTGTACTACTGTGCACGCTCTTACATCGACATGGACTATTGGGGTCAAGGCACCCTCGTAACGGTTTCTTCT 5D9_VLGAAATCGTGTTAACGCAGTCTCCAGGCACCCTGTCTTTGTCTCCAG 178GGGAAAGAGCCACCCTCTCTTGCAGGGCCAGTCAGAGTGTTAGCAGCAGCTACTTAGCCTGGTACCAGCAGAAACCTGGCCAGGCTCCCAGGCTCCTCATCTATGGAGCATCCAGCAGGGCCACTGGCATCCCAGACAGGTTCAGTGGCAGTGGATCCGGGACAGACTTCACTCTCACCATCAGCAGACTGGAGCCTGAAGATTTTGCAGTGTATTACTGTCAGCAGGATAACTGGAGCCCAACGTTCGGCCAGGGGACCAAAGTGGAAATCAAA 6B6_VHCAGGTGCAATTGGTTCAATCTGGTGCTGAAGTAAAAAAACCGGGCG 179CTTCCGTTAAAGTGAGCTGCAAAGCATCCGGATACACCTTCACTTCCTATTACATGCACTGGGTTCGTCAAGCCCCGGGCCAGGGTCTGGAATGGATGGGCATCATTAACCCAAGCGGTGGCTCTACCTCCTACGCGCAGAAATTCCAGGGTCGCGTCACGATGACCCGTGACACTAGCACCTCTACCGTTTATATGGAGCTGTCCAGCCTGCGTTCTGAAGATACTGCAGTGTACTACTGTGCACGCTCTTACGTTGACATGGACTATTGGGGTCAAGGCACCCTCGTAACGGTTTCTTCT 6B6_VLGAAATCGTGTTAACGCAGTCTCCAGGCACCCTGTCTTTGTCTCCAG 180GGGAAAGAGCCACCCTCTCTTGCAGGGCCAGTCAGAGTGTTAGCAGCAGCTACCTAGCCTGGTACCAGCAGAAACCTGGCCAGGCTCCCAGGCTCCTCATCTATGGAGCATCCAGCAGGGCCACTGGCATCCCAGACAGGTTCAGTGGCAGTGGATCCGGGACAGACTTCACTCTCACCATCAGCAGACTGGAGCCTGAAGATTTTGCAGTGTATTACTGTCAGCAGGATATTTGGAGCCCAACGTTCGGCCAGGGGACCAAAGTGGAAATCAAA 14E4_VHGAGGTGCAATTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGG 181GGTCCCTGAGACTCTCCTGTGCAGCCTCCGGATTCACCTTTAGCAGTTATGCCATGAGCTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTCTCAGCTATTAGTGGTAGTGGTGGTAGCACATACTACGCAGACTCCGTGAAGGGCCGGTTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTATCTGCAGATGAACAGCCTGAGAGCCGAGGACACGGCCGTATATTACTGTGCGAAAGACTCTTCTTACGTTGAATGGTACGCTTTCGACTACTGGGGCCAAGGAACCCTGGTCACCGTCTCGAGT 14E4_VLGAAATCGTGTTAACGCAGTCTCCAGGCACCCTGTCTTTGTCTCCAG 182GGGAAAGAGCCACCCTCTCTTGCAGGGCCAGTCAGAGTGTTAGCAGCAGCTACTTAGCCTGGTACCAGCAGAAACCTGGCCAGGCTCCCAGGCTCCTCATCTATGGAGCATCCAGCAGGGCCACTGGCATCCCAGACAGGTTCAGTGGCAGTGGATCCGGGACAGACTCCACTCTCACCATCAGCAGACTGGAGCCTGAAGATTTTGCAGTGTATTACTGTCAGCAGCCAACCAGCAGCCCAATTACGTTCGGCCAGGGGACCAAAGTGGAAATCA AA CD3 heavy GAGGTGCAGCTGCTGGAATCTGGCGGCGGACTGGTGCAGCCT 183 chainGGCGGATCTCTGAGACTGAGCTGTGCCGCCAGCGGCTTCACC (VHCH1)TTCAGCACCTACGCCATGAACTGGGTGCGCCAGGCCCCTGGCAAAGGCCTGGAATGGGTGTCCCGGATCAGAAGCAAGTACAACAACTACGCCACCTACTACGCCGACAGCGTGAAGGGCCGGTTCACCATCAGCCGGGACGACAGCAAGAACACCCTGTACCTGCAGATGAACAGCCTGCGGGCCGAGGACACCGCCGTGTACTATTGTGTGCGGCACGGCAACTTCGGCAACAGCTATGTGTCTTGGTTTGCCTACTGGGGCCAGGGCACCCTCGTGACCGTGTCAAGCGCTAGTACCAAGGGCCCCAGCGTGTTCCCCCTGGCACCCAGCAGCAAGAGCACATCTGGCGGAACAGCCGCTCTGGGCTGTCTGGTGAAAGACTACTTCCCCGAGCCCGTGACCGTGTCTTGGAACTCTGGCGCCCTGACCAGCGGCGTGCACACCTTTCCAGCCGTGCTGCAGAGCAGCGGCCTGTACTCCCTGTCCTCCGTGGTCACCGTGCCCTCTAGCTCCCTGGGAACACAGACATATATCTGTAATGTCAATCACAAGCCTTCCAACACCAAAGTCGATAAGAAAGTCGAG CCCAAGAGCTGC CrossedGAGGTGCAGCTGCTGGAATCTGGCGGCGGACTGGTGCAGCCTGGCG 184 CD3 heavyGATCTCTGAGACTGAGCTGTGCCGCCAGCGGCTTCACCTTCAGCAC chainCTACGCCATGAACTGGGTGCGCCAGGCCCCTGGCAAAGGCCTGGAA (VHCκ)TGGGTGTCCCGGATCAGAAGCAAGTACAACAACTACGCCACCTACTACGCCGACAGCGTGAAGGGCCGGTTCACCATCAGCCGGGACGACAGCAAGAACACCCTGTACCTGCAGATGAACAGCCTGCGGGCCGAGGACACCGCCGTGTACTATTGTGTGCGGCACGGCAACTTCGGCAACAGCTATGTGTCTTGGTTTGCCTACTGGGGCCAGGGCACCCTCGTGACCGTGTCAAGCGCTAGTGTGGCCGCTCCCTCCGTGTTTATCTTTCCCCCATCCGATGAACAGCTGAAAAGCGGCACCGCCTCCGTCGTGTGTCTGCTGAACAATTTTTACCCTAGGGAAGCTAAAGTGCAGTGGAAAGTGGATAACGCACTGCAGTCCGGCAACTCCCAGGAATCTGTGACAGAACAGGACTCCAAGGACAGCACCTACTCCCTGTCCTCCACCCTGACACTGTCTAAGGCTGATTATGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGA GAGTGT MutagenesisGCAGGCAAGCATTATGCAGCGGACTTTTGGTCAAGG 185 primer GAB7734 N95Q MutagenesisCAGGCAAGCATTATGAGCCGGACTTTTGGTCAAGG 186 primer GAB7735 N95S MutagenesisCATTATGAACCGGGCTTTTGGTCAAGGCACCAAGGTC 187 primer GAB7736 T97AMutagenesis CATTATGAACCGGAATTTTGGTCAAGGCACCAAGGTC 188 primer GAB7737T97N VHCH1[16D5]_ GAGGTGCAATTGGTTGAATCTGGTGGTGGTCTGGTAAAACCGGGCG 189VHCH1 GTTCCCTGCGTCTGAGCTGCGCGGCTTCCGGATTCACCTTCTCCAA [CD3]_CGCGTGGATGAGCTGGGTTCGCCAGGCCCCGGGCAAAGGCCTCGAG Fcknob_TGGGTTGGTCGTATCAAGTCTAAAACTGACGGTGGCACCACGGATT PGLALAACGCGGCTCCAGTTAAAGGTCGTTTTACCATTTCCCGCGACGATAG pCON999CAAAAACACTCTGTATCTGCAGATGAACTCTCTGAAAACTGAAGAC (InvertedACCGCAGTCTACTACTGTACTACCCCGTGGGAATGGTCTTGGTACG TCB withATTATTGGGGCCAGGGCACGCTGGTTACGGTGTCTTCCGCTAGCAC 16D5 2 + 1:AAAGGGCCCTAGCGTGTTCCCTCTGGCCCCCAGCAGCAAGAGCACA pCON999 +AGCGGCGGAACAGCCGCCCTGGGCTGCCTCGTGAAGGACTACTTCC pCON983 +CCGAGCCCGTGACAGTGTCTTGGAACAGCGGAGCCCTGACAAGCGG pETR13197)CGTGCACACTTTCCCTGCCGTGCTGCAGAGCAGCGGCCTGTACTCCCTGAGCAGCGTGGTCACCGTGCCTAGCAGCAGCCTGGGCACCCAGACCTACATCTGCAACGTGAACCACAAGCCCAGCAACACCAAAGTGGACAAGAAGGTGGAGCCCAAGAGCTGTGATGGCGGAGGAGGGTCCGGAGGCGGAGGATCCGAGGTGCAGCTGCTGGAATCTGGCGGCGGACTGGTGCAGCCTGGCGGATCTCTGAGACTGAGCTGTGCCGCCAGCGGCTTCACCTTCAGCACCTACGCCATGAACTGGGTGCGCCAGGCCCCTGGCAAAGGCCTGGAATGGGTGTCCCGGATCAGAAGCAAGTACAACAACTACGCCACCTACTACGCCGACAGCGTGAAGGGCCGGTTCACCATCAGCCGGGACGACAGCAAGAACACCCTGTACCTGCAGATGAACAGCCTGCGGGCCGAGGACACCGCCGTGTACTATTGTGTGCGGCACGGCAACTTCGGCAACAGCTATGTGTCTTGGTTTGCCTACTGGGGCCAGGGCACCCTCGTGACCGTGTCAAGCGCTAGTACCAAGGGCCCCAGCGTGTTCCCCCTGGCACCCAGCAGCAAGAGCACATCTGGCGGAACAGCCGCTCTGGGCTGTCTGGTGAAAGACTACTTCCCCGAGCCCGTGACCGTGTCTTGGAACTCTGGCGCCCTGACCAGCGGCGTGCACACCTTTCCAGCCGTGCTGCAGAGCAGCGGCCTGTACTCCCTGTCCTCCGTGGTCACCGTGCCCTCTAGCTCCCTGGGAACACAGACATATATCTGTAATGTCAATCACAAGCCTTCCAACACCAAAGTCGATAAGAAAGTCGAGCCCAAGAGCTGCGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAAGCTGCAGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCGGCGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATGCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTGTGGTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAA VHCH1[16D5]_GAGGTGCAATTGGTTGAATCTGGTGGTGGTCTGGTAAAACCGGGCG 190 Fchole_GTTCCCTGCGTCTGAGCTGCGCGGCTTCCGGATTCACCTTCTCCAA PGLALA_CGCGTGGATGAGCTGGGTTCGCCAGGCCCCGGGCAAAGGCCTCGAG HYRFTGGGTTGGTCGTATCAAGTCTAAAACTGACGGTGGCACCACGGATT pCON983ACGCGGCTCCAGTTAAAGGTCGTTTTACCATTTCCCGCGACGATAGCAAAAACACTCTGTATCTGCAGATGAACTCTCTGAAAACTGAAGACACCGCAGTCTACTACTGTACTACCCCGTGGGAATGGTCTTGGTACGATTATTGGGGCCAGGGCACGCTGGTTACGGTGTCTTCCGCTAGCACCAAGGGCCCCTCCGTGTTCCCCCTGGCCCCCAGCAGCAAGAGCACCAGCGGCGGCACAGCCGCTCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAGCCCGTGACCGTGTCCTGGAACAGCGGAGCCCTGACCTCCGGCGTGCACACCTTCCCCGCCGTGCTGCAGAGTTCTGGCCTGTATAGCCTGAGCAGCGTGGTCACCGTGCCTTCTAGCAGCCTGGGCACCCAGACCTACATCTGCAACGTGAACCACAAGCCCAGCAACACCAAGGTGGACAAGAAGGTGGAGCCCAAGAGCTGCGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAAGCTGCAGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCGGCGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTGCACCCTGCCCCCATCCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTCTCGTGCGCAGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCGTGAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCGCTTCACGCAGAAGAGCCTCTC CCTGTCTCCGGGTAAACD3_common CAGGCCGTCGTGACCCAGGAACCCAGCCTGACAGTGTCTCCTGGCG 191 lightGCACCGTGACCCTGACATGTGGCAGTTCTACAGGCGCCGTGACCAC chainCAGCAACTACGCCAACTGGGTGCAGGAAAAGCCCGGCCAGGCCTTC pETR13197AGAGGACTGATCGGCGGCACCAACAAGAGAGCCCCTGGCACCCCTGCCAGATTCAGCGGATCTCTGCTGGGAGGAAAGGCCGCCCTGACACTGTCTGGCGCCCAGCCAGAAGATGAGGCCGAGTACTACTGCGCCCTGTGGTACAGCAACCTGTGGGTGTTCGGCGGAGGCACCAAGCTGACAGTCCTAGGTCAACCCAAGGCTGCCCCCAGCGTGACCCTGTTCCCCCCCAGCAGCGAGGAACTGCAGGCCAACAAGGCCACCCTGGTCTGCCTGATCAGCGACTTCTACCCAGGCGCCGTGACCGTGGCCTGGAAGGCCGACAGCAGCCCCGTGAAGGCCGGCGTGGAGACCACCACCCCCAGCAAGCAGAGCAACAACAAGTACGCCGCCAGCAGCTACCTGAGCCTGACCCCCGAGCAGTGGAAGAGCCACAGGTCCTACAGCTGCCAGGTGACCCACGAGGGCAGCACCGTGGAGAAAACCGTGGCCCCCACCGAGTGCAG C VHCH1[CD3]_GAGGTGCAGCTGCTGGAATCTGGCGGCGGACTGGTGCAGCCTGGCG 192 VHCH1[16D5]_GATCTCTGAGACTGAGCTGTGCCGCCAGCGGCTTCACCTTCAGCAC Fcknob_CTACGCCATGAACTGGGTGCGCCAGGCCCCTGGCAAAGGCCTGGAA PGLALATGGGTGTCCCGGATCAGAAGCAAGTACAACAACTACGCCACCTACT pETR13932ACGCCGACAGCGTGAAGGGCCGGTTCACCATCAGCCGGGACGACAG (ClassicalCAAGAACACCCTGTACCTGCAGATGAACAGCCTGCGGGCCGAGGAC TCB withACCGCCGTGTACTATTGTGTGCGGCACGGCAACTTCGGCAACAGCT 16D5;2 + 1:ATGTGTCTTGGTTTGCCTACTGGGGCCAGGGCACCCTCGTGACCGT pETR13932 +GTCATCTGCTAGCACAAAGGGCCCTAGCGTGTTCCCTCTGGCCCCC pCON983 +AGCAGCAAGAGCACAAGCGGCGGAACAGCCGCCCTGGGCTGCCTCG pETR13197)TGAAGGACTACTTCCCCGAGCCCGTGACAGTGTCTTGGAACAGCGGAGCCCTGACAAGCGGCGTGCACACCTTCCCTGCCGTGCTGCAGAGCAGCGGCCTGTACTCCCTGAGCAGCGTGGTCACCGTGCCTAGCAGCAGCCTGGGCACCCAGACCTACATCTGCAACGTGAACCACAAGCCCAGCAACACCAAAGTGGACAAGAAGGTGGAGCCCAAGAGCTGTGATGGCGGAGGAGGGTCCGGAGGCGGAGGATCCGAGGTGCAATTGGTTGAATCTGGTGGTGGTCTGGTAAAACCGGGCGGTTCCCTGCGTCTGAGCTGCGCGGCTTCCGGATTCACCTTCTCCAACGCGTGGATGAGCTGGGTTCGCCAGGCCCCGGGCAAAGGCCTCGAGTGGGTTGGTCGTATCAAGTCTAAAACTGACGGTGGCACCACGGATTACGCGGCTCCAGTTAAAGGTCGTTTTACCATTTCCCGCGACGATAGCAAAAACACTCTGTATCTGCAGATGAACTCTCTGAAAACTGAAGACACCGCAGTCTACTACTGTACTACCCCGTGGGAATGGTCTTGGTACGATTATTGGGGCCAGGGCACGCTGGTTACGGTGTCTAGCGCTAGTACCAAGGGCCCCAGCGTGTTCCCCCTGGCACCCAGCAGCAAGAGCACATCTGGCGGAACAGCCGCTCTGGGCTGTCTGGTGAAAGACTACTTCCCCGAGCCCGTGACCGTGTCTTGGAACTCTGGCGCCCTGACCAGCGGCGTGCACACCTTTCCAGCCGTGCTGCAGAGCAGCGGCCTGTACTCCCTGTCCTCCGTGGTCACCGTGCCCTCTAGCTCCCTGGGAACACAGACATATATCTGTAATGTCAATCACAAGCCTTCCAACACCAAAGTCGATAAGAAAGTCGAGCCCAAGAGCTGCGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAAGCTGCAGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCGGCGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATGCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTGTGGTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAA VHCH1[CD3]_GAGGTGCAGCTGCTGGAATCTGGCGGCGGACTGGTGCAGCCTGGCG 193 Fcknob_GATCTCTGAGACTGAGCTGTGCCGCCAGCGGCTTCACCTTCAGCAC PGLALACTACGCCATGAACTGGGTGCGCCAGGCCCCTGGCAAAGGCCTGGAA pETR13719TGGGTGTCCCGGATCAGAAGCAAGTACAACAACTACGCCACCTACT (16D5 IgGACGCCGACAGCGTGAAGGGCCGGTTCACCATCAGCCGGGACGACAG format 1 + 1: CAAGAACACCCTGTACCTGCAGATGAACAGCCTGCGGGCCGAGGAC pETR13719 +ACCGCCGTGTACTATTGTGTGCGGCACGGCAACTTCGGCAACAGCT pCON983 +ATGTGTCTTGGTTTGCCTACTGGGGCCAGGGCACCCTCGTGACCGT pETR13197)GTCATCTGCTAGCACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAAAGTTGAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAAGCTGCAGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCGGCGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATGCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTGTGGTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAA Fc_hole_GACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAACTCCTGG 194 PGLALA_HYRFGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCT pETR10755CATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTG (16D5 Head-AGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCG to 1 + 1:TGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAA pCON999 +CAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGAC pETR10755 +TGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCC pETR13197)TCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTGCACCCTGCCCCCATCCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTCTCGTGCGCAGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCGTGAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCGCTTCACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAA VHCH1[9D11]_CAGGTGCAATTGGTTCAATCTGGTGCTGAAGTAAAAAAACCGGGCG 195 VHCL[CD3]_CTTCCGTTAAAGTGAGCTGCAAAGCATCCGGATACACCTTCACTTC Fcknob_CTATTACATGCACTGGGTTCGTCAAGCCCCGGGCCAGGGTCTGGAA PGLALATGGATGGGCATCATTAACCCAAGCGGTGGCCCTACCTCCTACGCGC pCON1057AGAAATTCCAGGGTCGCGTCACGATGACCCGTGACACTAGCACCTC (9D11TACCGTTTATATGGAGCTGTCCAGCCTGCGTTCTGAAGATACTGCA invertedGTGTACTACTGTGCACGCGGTGACTTCGCTTGGCTGGACTATTGGG format,GTCAAGGCACCCTCGTAACGGTTTCTTCTGCTAGCACAAAGGGCCC 2 + 1:CAGCGTGTTCCCTCTGGCCCCTAGCAGCAAGAGCACATCTGGCGGA pCON1057 +ACAGCCGCCCTGGGCTGCCTCGTGAAGGACTACTTTCCCGAGCCTG pCON1051 +TGACCGTGTCCTGGAACTCTGGCGCCCTGACAAGCGGCGTGCACAC pCON1063 +CTTTCCAGCCGTGCTGCAGAGCAGCGGCCTGTACTCTCTGAGCAGC pETR12940)GTGGTCACCGTGCCTAGCAGCAGCCTGGGCACCCAGACCTACATCTGCAACGTGAACCACAAGCCCAGCAACACCAAAGTGGACAAGAAGGTGGAGCCCAAGAGCTGTGATGGCGGAGGAGGGTCCGGAGGCGGAGGATCCGAGGTGCAGCTGCTGGAATCTGGCGGCGGACTGGTGCAGCCTGGCGGATCTCTGAGACTGAGCTGTGCCGCCAGCGGCTTCACCTTCAGCACCTACGCCATGAACTGGGTGCGCCAGGCCCCTGGCAAAGGCCTGGAATGGGTGTCCCGGATCAGAAGCAAGTACAACAACTACGCCACCTACTACGCCGACAGCGTGAAGGGCCGGTTCACCATCAGCCGGGACGACAGCAAGAACACCCTGTACCTGCAGATGAACAGCCTGCGGGCCGAGGACACCGCCGTGTACTATTGTGTGCGGCACGGCAACTTCGGCAACAGCTATGTGTCTTGGTTTGCCTACTGGGGCCAGGGCACCCTCGTGACCGTGTCAAGCGCTAGTGTGGCCGCTCCCTCCGTGTTTATCTTTCCCCCATCCGATGAACAGCTGAAAAGCGGCACCGCCTCCGTCGTGTGTCTGCTGAACAATTTTTACCCTAGGGAAGCTAAAGTGCAGTGGAAAGTGGATAACGCACTGCAGTCCGGCAACTCCCAGGAATCTGTGACAGAACAGGACTCCAAGGACAGCACCTACTCCCTGTCCTCCACCCTGACACTGTCTAAGGCTGATTATGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGTGACAAGACCCACACCTGTCCCCCTTGTCCTGCCCCTGAAGCTGCTGGCGGCCCTTCTGTGTTCCTGTTCCCCCCAAAGCCCAAGGACACCCTGATGATCAGCCGGACCCCCGAAGTGACCTGCGTGGTGGTGGATGTGTCCCACGAGGACCCTGAAGTGAAGTTCAATTGGTACGTGGACGGCGTGGAAGTGCACAACGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCGGCGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATGCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTGTGGTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAA 9D11_CAGGTGCAATTGGTTCAATCTGGTGCTGAAGTAAAAAAACCGGGCG 196 Fchole_CTTCCGTTAAAGTGAGCTGCAAAGCATCCGGATACACCTTCACTTC PGLALA_HYRFCTATTACATGCACTGGGTTCGTCAAGCCCCGGGCCAGGGTCTGGAA pCON1051TGGATGGGCATCATTAACCCAAGCGGTGGCCCTACCTCCTACGCGCAGAAATTCCAGGGTCGCGTCACGATGACCCGTGACACTAGCACCTCTACCGTTTATATGGAGCTGTCCAGCCTGCGTTCTGAAGATACTGCAGTGTACTACTGTGCACGCGGTGACTTCGCTTGGCTGGACTATTGGGGTCAAGGCACCCTCGTAACGGTTTCTTCTGCTAGCACCAAGGGCCCCTCCGTGTTCCCCCTGGCCCCCAGCAGCAAGAGCACCAGCGGCGGCACAGCCGCTCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAGCCCGTGACCGTGTCCTGGAACAGCGGAGCCCTGACCTCCGGCGTGCACACCTTCCCCGCCGTGCTGCAGAGTTCTGGCCTGTATAGCCTGAGCAGCGTGGTCACCGTGCCTTCTAGCAGCCTGGGCACCCAGACCTACATCTGCAACGTGAACCACAAGCCCAGCAACACCAAGGTGGACAAGAAGGTGGAGCCCAAGAGCTGCGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAAGCTGCAGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCGGCGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTGCACCCTGCCCCCATCCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTCTCGTGCGCAGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCGTGAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCC GGGTAAA 9D11_LCGATATTGTTATGACTCAATCTCCACTGTCTCTGCCGGTGACTCCAG 197 pCON1063GCGAACCGGCGAGCATTTCTTGCCGTTCCAGCCAGTCTCTGCTGCACTCCAACGGCTACAACTATCTCGATTGGTACCTGCAAAAACCGGGTCAGAGCCCTCAGCTGCTGATCTACCTGGGCTCTAACCGCGCTTCCGGTGTACCGGACCGTTTCAGCGGCTCTGGATCCGGCACCGATTTCACGTTGAAAATCAGCCGTGTTGAAGCAGAAGACGTGGGCGTTTATTACTGTATGCAGGCAAGCATTATGAACCGGACTTTTGGTCAAGGCACCAAGGTCGAAATTAAACGTACGGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAA CAGGGGAGAGTGT VLCH1[CD3]CAGGCCGTCGTGACCCAGGAACCCAGCCTGACAGTGTCTCCTGGCG 198 pETR12940GCACCGTGACCCTGACATGTGGCAGTTCTACAGGCGCCGTGACCACCAGCAACTACGCCAACTGGGTGCAGGAAAAGCCCGGCCAGGCCTTCAGAGGACTGATCGGCGGCACCAACAAGAGAGCCCCTGGCACCCCTGCCAGATTCAGCGGATCTCTGCTGGGAGGAAAGGCCGCCCTGACACTGTCTGGCGCCCAGCCAGAAGATGAGGCCGAGTACTACTGCGCCCTGTGGTACAGCAACCTGTGGGTGTTCGGCGGAGGCACCAAGCTGACAGTGCTGAGCAGCGCTTCCACCAAAGGCCCTTCCGTGTTTCCTCTGGCTCCTAGCTCCAAGTCCACCTCTGGAGGCACCGCTGCTCTCGGATGCCTCGTGAAGGATTATTTTCCTGAGCCTGTGACAGTGTCCTGGAATAGCGGAGCACTGACCTCTGGAGTGCATACTTTCCCCGCTGTGCTGCAGTCCTCTGGACTGTACAGCCTGAGCAGCGTGGTGACAGTGCCCAGCAGCAGCCTGGGCACCCAGACCTACATCTGCAACGTGAACCACAAGCCCAGCAACACCAAGGTGGACAAGAAGGTGGAACCCAAGTCTTGT VHCL[CD3]_GAGGTGCAGCTGCTGGAATCTGGCGGCGGACTGGTGCAGCCTGGCG 199 Fcknob_GATCTCTGAGACTGAGCTGTGCCGCCAGCGGCTTCACCTTCAGCAC PGLALACTACGCCATGAACTGGGTGCGCCAGGCCCCTGGCAAAGGCCTGGAA pETR13378TGGGTGTCCCGGATCAGAAGCAAGTACAACAACTACGCCACCTACT (9D11ACGCCGACAGCGTGAAGGGCCGGTTCACCATCAGCCGGGACGACAG CrossMabCAAGAACACCCTGTACCTGCAGATGAACAGCCTGCGGGCCGAGGAC format, ACCGCCGTGTACTATTGTGTGCGGCACGGCAACTTCGGCAACAGCT 1 + 1:ATGTGTCTTGGTTTGCCTACTGGGGCCAGGGCACCCTCGTGACCGT pETR13378 +GTCATCTGCTAGCGTGGCCGCTCCCTCCGTGTTTATCTTTCCCCCA pCON1051 +TCCGATGAACAGCTGAAAAGCGGCACCGCCTCCGTCGTGTGTCTGC pCON1063 +TGAACAATTTTTACCCTAGGGAAGCTAAAGTGCAGTGGAAAGTGGA pETR12940)TAACGCACTGCAGTCCGGCAACTCCCAGGAATCTGTGACAGAACAGGACTCCAAGGACAGCACCTACTCCCTGTCCTCCACCCTGACACTGTCTAAGGCTGATTATGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGTGACAAGACCCACACCTGTCCCCCTTGTCCTGCCCCTGAAGCTGCTGGCGGCCCTTCTGTGTTCCTGTTCCCCCCAAAGCCCAAGGACACCCTGATGATCAGCCGGACCCCCGAAGTGACCTGCGTGGTGGTGGATGTGTCCCACGAGGACCCTGAAGTGAAGTTCAATTGGTACGTGGACGGCGTGGAAGTGCACAACGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCGGCGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATGCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTGTGGTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAA 16D5GAGGTGCAATTGGTTGAATCTGGTGGTGGTCTGGTAAAACCGGGCG 200 invertedGTTCCCTGCGTCTGAGCTGCGCGGCTTCCGGATTCACCTTCTCCAA 2 + 1 withCGCGTGGATGAGCTGGGTTCGCCAGGCCCCGGGCAAAGGCCTCGAG N100A inTGGGTTGGTCGTATCAAGTCTAAAACTGACGGTGGCACCACGGATT CDR H3ACGCGGCTCCAGTTAAAGGTCGTTTTACCATTTCCCGCGACGATAG pETR14096CAAAAACACTCTGTATCTGCAGATGAACTCTCTGAAAACTGAAGAC (pETR14096 +ACCGCAGTCTACTACTGTACTACCCCGTGGGAATGGTCTTGGTACG pCON983 +ATTATTGGGGCCAGGGCACGCTGGTTACGGTGTCTTCCGCTAGCAC pETR13197)AAAGGGCCCTAGCGTGTTCCCTCTGGCCCCCAGCAGCAAGAGCACAAGCGGCGGAACAGCCGCCCTGGGCTGCCTCGTGAAGGACTACTTCCCCGAGCCCGTGACAGTGTCTTGGAACAGCGGAGCCCTGACAAGCGGCGTGCACACTTTCCCTGCCGTGCTGCAGAGCAGCGGCCTGTACTCCCTGAGCAGCGTGGTCACCGTGCCTAGCAGCAGCCTGGGCACCCAGACCTACATCTGCAACGTGAACCACAAGCCCAGCAACACCAAAGTGGACAAGAAGGTGGAGCCCAAGAGCTGTGATGGCGGAGGAGGGTCCGGAGGCGGAGGATCCGAGGTGCAGCTGCTGGAATCTGGCGGCGGACTGGTGCAGCCTGGCGGATCTCTGAGACTGAGCTGTGCCGCCAGCGGCTTCACCTTCAGCACCTACGCCATGAACTGGGTGCGCCAGGCCCCTGGCAAAGGCCTGGAATGGGTGTCCCGGATCAGAAGCAAGTACAACAACTACGCCACCTACTACGCCGACAGCGTGAAGGGCCGGTTCACCATCAGCCGGGACGACAGCAAGAACACCCTGTACCTGCAGATGAACAGCCTGCGGGCCGAGGACACCGCCGTGTACTATTGTGTGCGGCACGGCAACTTCGGCGCCAGCTATGTGTCTTGGTTTGCCTACTGGGGCCAGGGCACCCTCGTGACCGTGTCAAGCGCTAGTACCAAGGGCCCCAGCGTGTTCCCCCTGGCACCCAGCAGCAAGAGCACATCTGGCGGAACAGCCGCTCTGGGCTGTCTGGTGAAAGACTACTTCCCCGAGCCCGTGACCGTGTCTTGGAACTCTGGCGCCCTGACCAGCGGCGTGCACACCTTTCCAGCCGTGCTGCAGAGCAGCGGCCTGTACTCCCTGTCCTCCGTGGTCACCGTGCCCTCTAGCTCCCTGGGAACACAGACATATATCTGTAATGTCAATCACAAGCCTTCCAACACCAAAGTCGATAAGAAAGTCGAGCCCAAGAGCTGCGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAAGCTGCAGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCGGCGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATGCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTGTGGTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAA 16D5GAGGTGCAATTGGTTGAATCTGGTGGTGGTCTGGTAAAACCGGGCG 201 invertedGTTCCCTGCGTCTGAGCTGCGCGGCTTCCGGATTCACCTTCTCCAA 2 + 1 withCGCGTGGATGAGCTGGGTTCGCCAGGCCCCGGGCAAAGGCCTCGAG S100aA inTGGGTTGGTCGTATCAAGTCTAAAACTGACGGTGGCACCACGGATT CDR H3ACGCGGCTCCAGTTAAAGGTCGTTTTACCATTTCCCGCGACGATAG pETR14097CAAAAACACTCTGTATCTGCAGATGAACTCTCTGAAAACTGAAGAC (pETR14097 +ACCGCAGTCTACTACTGTACTACCCCGTGGGAATGGTCTTGGTACG pCON983 +ATTATTGGGGCCAGGGCACGCTGGTTACGGTGTCTTCCGCTAGCAC pETR13197)AAAGGGCCCTAGCGTGTTCCCTCTGGCCCCCAGCAGCAAGAGCACAAGCGGCGGAACAGCCGCCCTGGGCTGCCTCGTGAAGGACTACTTCCCCGAGCCCGTGACAGTGTCTTGGAACAGCGGAGCCCTGACAAGCGGCGTGCACACTTTCCCTGCCGTGCTGCAGAGCAGCGGCCTGTACTCCCTGAGCAGCGTGGTCACCGTGCCTAGCAGCAGCCTGGGCACCCAGACCTACATCTGCAACGTGAACCACAAGCCCAGCAACACCAAAGTGGACAAGAAGGTGGAGCCCAAGAGCTGTGATGGCGGAGGAGGGTCCGGAGGCGGAGGATCCGAGGTGCAGCTGCTGGAATCTGGCGGCGGACTGGTGCAGCCTGGCGGATCTCTGAGACTGAGCTGTGCCGCCAGCGGCTTCACCTTCAGCACCTACGCCATGAACTGGGTGCGCCAGGCCCCTGGCAAAGGCCTGGAATGGGTGTCCCGGATCAGAAGCAAGTACAACAACTACGCCACCTACTACGCCGACAGCGTGAAGGGCCGGTTCACCATCAGCCGGGACGACAGCAAGAACACCCTGTACCTGCAGATGAACAGCCTGCGGGCCGAGGACACCGCCGTGTACTATTGTGTGCGGCACGGCAACTTCGGCAACGCCTATGTGTCTTGGTTTGCCTACTGGGGCCAGGGCACCCTCGTGACCGTGTCAAGCGCTAGTACCAAGGGCCCCAGCGTGTTCCCCCTGGCACCCAGCAGCAAGAGCACATCTGGCGGAACAGCCGCTCTGGGCTGTCTGGTGAAAGACTACTTCCCCGAGCCCGTGACCGTGTCTTGGAACTCTGGCGCCCTGACCAGCGGCGTGCACACCTTTCCAGCCGTGCTGCAGAGCAGCGGCCTGTACTCCCTGTCCTCCGTGGTCACCGTGCCCTCTAGCTCCCTGGGAACACAGACATATATCTGTAATGTCAATCACAAGCCTTCCAACACCAAAGTCGATAAGAAAGTCGAGCCCAAGAGCTGCGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAAGCTGCAGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCGGCGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATGCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTGTGGTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAA CD3 lightCAGGCCGTCGTGACCCAGGAACCCAGCCTGACAGTGTCTCCTGGCG 202 chain fusedGCACCGTGACCCTGACATGTGGCAGTTCTACAGGCGCCGTGACCAC to CH1;CAGCAACTACGCCAACTGGGTGCAGGAAAAGCCCGGCCAGGCCTTC Fc_PGLALA;AGAGGACTGATCGGCGGCACCAACAAGAGAGCCCCTGGCACCCCTG pETR13862CCAGATTCAGCGGATCTCTGCTGGGAGGAAAGGCCGCCCTGACACT (Kappa-GTCTGGCGCCCAGCCAGAAGATGAGGCCGAGTACTACTGCGCCCTG lambdaTGGTACAGCAACCTGTGGGTGTTCGGCGGAGGCACCAAGCTGACAG antibody withTGCTGAGCAGCGCTAGCACCAAGGGCCCATCGGTCTTCCCCCTGGC CD3 commonACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGC light chainCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACT fused to CAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACA CH1 +GTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCC Fc_PGLALA.AGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGC VHs fused toCCAGCAACACCAAGGTGGACAAGAAAGTTGAGCCCAAATCTTGTGA kappa orCAAAACTCACACATGCCCACCGTGCCCAGCACCTGAAGCTGCAGGG lambdaGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCA constantTGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAG chainCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTG pETR13859 +GAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACA pETR13860 +GCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTG pETR13862)GCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCGGCGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCC GAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAA 16D5 VHGAGGTGCAATTGGTTGAATCTGGTGGTGGTCTGGTAAAACCGGGCG 203 fused toGTTCCCTGCGTCTGAGCTGCGCGGCTTCCGGATTCACCTTCTCCAA constantCGCGTGGATGAGCTGGGTTCGCCAGGCCCCGGGCAAAGGCCTCGAG kappaTGGGTTGGTCGTATCAAGTCTAAAACTGACGGTGGCACCACGGATT chain;ACGCGGCTCCAGTTAAAGGTCGTTTTACCATTTCCCGCGACGATAG pETR13859CAAAAACACTCTGTATCTGCAGATGAACTCTCTGAAAACTGAAGACACCGCAGTCTACTACTGTACTACCCCGTGGGAATGGTCTTGGTACGATTATTGGGGCCAGGGCACGCTGGTTACGGTGTCTTCCGCTAGCGTGGCCGCTCCCTCCGTGTTCATCTTCCCACCTTCCGACGAGCAGCTGAAGTCCGGCACCGCTTCTGTCGTGTGCCTGCTGAACAACTTCTACCCCCGCGAGGCCAAGGTGCAGTGGAAGGTGGACAACGCCCTGCAGTCCGGCAACAGCCAGGAATCCGTGACCGAGCAGGACTCCAAGGACAGCACCTACTCCCTGTCCTCCACCCTGACCCTGTCCAAGGCCGACTACGAGAAGCACAAGGTGTACGCCTGCGAAGTGACCCACCAGGGCCTGTCTAGCCCCGTGACCAAGTCTTTCAACCGGGGCGAGTGC CD3 VHGAAGTGCAGCTGCTGGAATCCGGCGGAGGACTGGTGCAGCCTGGCG 204 fused toGATCTCTGAGACTGTCTTGTGCCGCCTCCGGCTTCACCTTCTCCAC constantCTACGCCATGAACTGGGTGCGACAGGCTCCTGGCAAGGGCCTGGAA lambdaTGGGTGTCCCGGATCAGATCCAAGTACAACAACTACGCCACCTACT chain;ACGCCGACTCCGTGAAGGGCCGGTTCACCATCTCTCGGGACGACTC pETR13860CAAGAACACCCTGTACCTGCAGATGAACTCCCTGCGGGCCGAGGACACCGCCGTGTACTATTGTGTGCGGCACGGCAACTTCGGCAACTCCTATGTGTCTTGGTTTGCCTACTGGGGCCAGGGCACCCTCGTGACCGTGTCATCTGCTAGCCCCAAGGCTGCCCCCAGCGTGACCCTGTTTCCCCCCAGCAGCGAGGAACTGCAGGCCAACAAGGCCACCCTGGTCTGCCTGATCAGCGACTTCTACCCAGGCGCCGTGACCGTGGCCTGGAAGGCCGACAGCAGCCCCGTGAAGGCCGGCGTGGAGACCACCACCCCCAGCAAGCAGAGCAACAACAAGTACGCCGCCAGCAGCTACCTGAGCCTGACCCCCGAGCAGTGGAAGAGCCACAGGTCCTACAGCTGCCAGGTGAC CCACGAGGGCAGCACCGTGGAGAAAACCGTGGCCCCCACCGAGTGCAGC VHCH1[36F2]_CAGGTGCAATTGGTTCAATCTGGTGCTGAAGTAAAAAAACCGGGCGCTTCC 246 VHCL[CD3]_GTTAAAGTGAGCTGCAAAGCATCCGGATACACCTTCACTTCCTATTACATG Fcknob_CACTGGGTTCGTCAAGCCCCGGGCCAGGGTCTGGAATGGATGGGCATCATT PGLALAAACCCAAGCGGTGGCTCTACCTCCTACGCGCAGAAATTCCAGGGTCGCGTC pCON1056ACGATGACCCATGACACTAGCACCTCTACCGTTTATATGGAGCTGTCCAGCCTGCGTTCTGAAGATACTGCAGTGTACTACTGTGCACGCTCTTTCTTCACTGGTTTCCATCTGGACTATTGGGGTCAAGGCACCCTCGTAACGGTTTCTTCTGCTAGCACAAAGGGCCCCAGCGTGTTCCCTCTGGCCCCTAGCAGCAAGAGCACATCTGGCGGAACAGCCGCCCTGGGCTGCCTCGTGAAGGACTACTTTCCCGAGCCTGTGACCGTGTCCTGGAACTCTGGCGCCCTGACAAGCGGCGTGCACACCTTTCCAGCCGTGCTGCAGAGCAGCGGCCTGTACTCTCTGAGCAGCGTGGTCACCGTGCCTAGCAGCAGCCTGGGCACCCAGACCTACATCTGCAACGTGAACCACAAGCCCAGCAACACCAAAGTGGACAAGAAGGTGGAGCCCAAGAGCTGTGATGGCGGAGGAGGGTCCGGAGGCGGAGGATCCGAGGTGCAGCTGCTGGAATCTGGCGGCGGACTGGTGCAGCCTGGCGGATCTCTGAGACTGAGCTGTGCCGCCAGCGGCTTCACCTTCAGCACCTACGCCATGAACTGGGTGCGCCAGGCCCCTGGCAAAGGCCTGGAATGGGTGTCCCGGATCAGAAGCAAGTACAACAACTACGCCACCTACTACGCCGACAGCGTGAAGGGCCGGTTCACCATCAGCCGGGACGACAGCAAGAACACCCTGTACCTGCAGATGAACAGCCTGCGGGCCGAGGACACCGCCGTGTACTATTGTGTGCGGCACGGCAACTTCGGCAACAGCTATGTGTCTTGGTTTGCCTACTGGGGCCAGGGCACCCTCGTGACCGTGTCAAGCGCTAGTGTGGCCGCTCCCTCCGTGTTTATCTTTCCCCCATCCGATGAACAGCTGAAAAGCGGCACCGCCTCCGTCGTGTGTCTGCTGAACAATTTTTACCCTAGGGAAGCTAAAGTGCAGTGGAAAGTGGATAACGCACTGCAGTCCGGCAACTCCCAGGAATCTGTGACAGAACAGGACTCCAAGGACAGCACCTACTCCCTGTCCTCCACCCTGACACTGTCTAAGGCTGATTATGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGTGACAAGACCCACACCTGTCCCCCTTGTCCTGCCCCTGAAGCTGCTGGCGGCCCTTCTGTGTTCCTGTTCCCCCCAAAGCCCAAGGACACCCTGATGATCAGCCGGACCCCCGAAGTGACCTGCGTGGTGGTGGATGTGTCCCACGAGGACCCTGAAGTGAAGTTCAATTGGTACGTGGACGGCGTGGAAGTGCACAACGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCGGCGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATGCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTGTGGTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAA 36F2-FcholeCAGGTGCAATTGGTTCAATCTGGTGCTGAAGTAAAAAAACCGGGCGCTTCC 247 PGLALAGTTAAAGTGAGCTGCAAAGCATCCGGATACACCTTCACTTCCTATTACATG pCON1050CACTGGGTTCGTCAAGCCCCGGGCCAGGGTCTGGAATGGATGGGCATCATTAACCCAAGCGGTGGCTCTACCTCCTACGCGCAGAAATTCCAGGGTCGCGTCACGATGACCCATGACACTAGCACCTCTACCGTTTATATGGAGCTGTCCAGCCTGCGTTCTGAAGATACTGCAGTGTACTACTGTGCACGCTCTTTCTTCACTGGTTTCCATCTGGACTATTGGGGTCAAGGCACCCTCGTAACGGTTTCTTCTGCTAGCACCAAGGGCCCCTCCGTGTTCCCCCTGGCCCCCAGCAGCAAGAGCACCAGCGGCGGCACAGCCGCTCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAGCCCGTGACCGTGTCCTGGAACAGCGGAGCCCTGACCTCCGGCGTGCACACCTTCCCCGCCGTGCTGCAGAGTTCTGGCCTGTATAGCCTGAGCAGCGTGGTCACCGTGCCTTCTAGCAGCCTGGGCACCCAGACCTACATCTGCAACGTGAACCACAAGCCCAGCAACACCAAGGTGGACAAGAAGGTGGAGCCCAAGAGCTGCGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAAGCTGCAGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCGGCGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTGCACCCTGCCCCCATCCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTCTCGTGCGCAGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCGTGAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAA 36F2 LCGAAATCGTGTTAACGCAGTCTCCAGGCACCCTGTCTTTGTCTCCAGGGGAA  97 pCON1062AGAGCCACCCTCTCTTGCAGGGCCAGTCAGAGTGTTAGCAGCAGCTACTTAGCCTGGTACCAGCAGAAACCTGGCCAGGCTCCCAGGCTCCTCATCTATGGAGCATCCAGCAGGGCCACTGGCATCCCnAGACAGGTTCAGTGGCAGTGGATCCGGGACAGACTTCACTCTCACCATCAGCAGACTGGAGCCTGAAGATTTTGCAGTGTATTACTGTCAGCAGTATACCAACGAACATTATTATACGTTCGGCCAGGGGACCAAAGTGGAAATCAAACGTACGGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCANGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGT CD3CAGGCCGTCGTGACCCAGGAACCCAGCCTGACAGTGTCTCCTGGCGGCACC 198 VLCH1GTGACCCTGACATGTGGCAGTTCTACAGGCGCCGTGACCACCAGCAACTAC pETR1294OGCCAACTGGGTGCAGGAAAAGCCCGGCCAGGCCTTCAGAGGACTGATCGGCGGCACCAACAAGAGAGCCCCTGGCACCCCTGCCAGATTCAGCGGATCTCTGCTGGGAGGAAAGGCCGCCCTGACACTGTCTGGCGCCCAGCCAGAAGATGAGGCCGAGTACTACTGCGCCCTGTGGTACAGCAACCTGTGGGTGTTCGGCGGAGGCACCAAGCTGACAGTGCTGAGCAGCGCTTCCACCAAAGGCCCTTCCGTGTTTCCTCTGGCTCCTAGCTCCAAGTCCACCTCTGGAGGCACCGCTGCTCTCGGATGCCTCGTGAAGGATTATTTTCCTGAGCCTGTGACAGTGTCCTGGAATAGCGGAGCACTGACCTCTGGAGTGCATACTTTCCCCGCTGTGCTGCAGTCCTCTGGACTGTACAGCCTGAGCAGCGTGGTGACAGTGCCCAGCAGCAGCCTGGGCACCCAGACCTACATCTGCAACGTGAACCACAAGCCCAGCAACACCAAGGTGGACAAGAAGGTGGAACCCAAGTCTTGT Seq Name Sequence ID No K53ACAGACCGTCGTGACCCAGGAACCCAGCCTGACAGTGTCTCCTGGCGGCACC 205 ntGTGACCCTGACATGTGGCAGTTCTACAGGCGCCGTGACCACCAGCAACTACGCCAACTGGGTGCAGCAGAAGCCAGGCCAGGCTCCCAGAGGACTGATCGGCGGCACCAACGCCAGAGCCCCTGGCACCCCTGCCAGATTCAGCGGATCTCTGCTGGGAGGAAAGGCCGCCCTGACACTGTCTGGCGTGCAGCCTGAAGATGAGGCCGAGTACTACTGCGCCCTGTGGTACAGCAACCTGTGGGTGTTCGGCGGAGGCACCAAGCTGACAGTCCTA S93ACAGACCGTCGTGACCCAGGAACCCAGCCTGACAGTGTCTCCTGGCGGCACC 206 ntGTGACCCTGACATGTGGCAGTTCTACAGGCGCCGTGACCACCAGCAACTACGCCAACTGGGTGCAGCAGAAGCCAGGCCAGGCTCCCAGAGGACTGATCGGCGGCACCAACAAGAGAGCCCCTGGCACCCCTGCCAGATTCAGCGGATCTCTGCTGGGAGGAAAGGCCGCCCTGACACTGTCTGGCGTGCAGCCTGAAGATGAGGCCGAGTACTACTGCGCCCTGTGGTACGCCAACCTGTGGGTGTTCGGCGGAGGCACCAAGCTGACAGTCCTA S35HGAGGTGCAATTGGTGGAAAGCGGAGGCGGCCTCGTGAAGCCTGGCGGATCT 207 ntCTGAGACTGAGCTGTGCCGCCAGCGGCTTCACCTTCAGCAACGCCTGGATGCACTGGGTGCGCCAGGCCCCTGGAAAAGGACTCGAGTGGGTGGGACGGATCAAGAGCAAGACCGATGGCGGCACCACCGACTATGCCGCCCCTGTGAAGGGCCGGTTCACCATCAGCAGGGACGACAGCAAGAACACCCTGTACCTGCAGATGAACAGCCTGAAAACCGAGGACACCGCCGTGTACTACTGCACCACCCCCTGGGAGTGGTCTTGGTACGACTATTGGGGCCAGGGCACCCTCGTGACCGTGTCC TCTGCTAGC G49SGAGGTGCAATTGGTGGAAAGCGGAGGCGGCCTCGTGAAGCCTGGCGGATCT 208 ntCTGAGACTGAGCTGTGCCGCCAGCGGCTTCACCTTCAGCAACGCCTGGATGAGCTGGGTGCGCCAGGCCCCTGGAAAAGGACTCGAGTGGGTGTCCCGGATCAAGAGCAAGACCGATGGCGGCACCACCGACTATGCCGCCCCTGTGAAGGGCCGGTTCACCATCAGCAGGGACGACAGCAAGAACACCCTGTACCTGCAGATGAACAGCCTGAAAACCGAGGACACCGCCGTGTACTACTGCACCACCCCCTGGGAGTGGTCTTGGTACGACTATTGGGGCCAGGGCACCCTCGTGACCGTGTCC TCTGCTAGC R50SGAGGTGCAATTGGTGGAAAGCGGAGGCGGCCTCGTGAAGCCTGGCGGATCT 209 ntCTGAGACTGAGCTGTGCCGCCAGCGGCTTCACCTTCAGCAACGCCTGGATGAGCTGGGTGCGCCAGGCCCCTGGAAAAGGACTCGAGTGGGTGGGATCTATCAAGAGCAAGACCGACGGCGGCACCACCGACTATGCCGCCCCTGTGAAGGGCCGGTTCACCATCAGCAGGGACGACAGCAAGAACACCCTGTACCTGCAGATGAACAGCCTGAAAACCGAGGACACCGCCGTGTACTACTGCACCACCCCCTGGGAGTGGTCTTGGTACGACTATTGGGGCCAGGGCACCCTCGTGACCGTGTCC TCT GCTAGC W96YGAGGTGCAATTGGTGGAAAGCGGAGGCGGCCTCGTGAAGCCTGGCGGATCT 210 ntCTGAGACTGAGCTGTGCCGCCAGCGGCTTCACCTTCAGCAACGCCTGGATGAGCTGGGTGCGCCAGGCCCCTGGAAAAGGACTCGAGTGGGTGGGACGGATCAAGAGCAAGACCGATGGCGGCACCACCGACTATGCCGCCCCTGTGAAGGGCCGGTTCACCATCAGCAGGGACGACAGCAAGAACACCCTGTACCTGCAGATGAACAGCCTGAAAACCGAGGACACCGCCGTGTACTACTGCACCACCCCCTACGAGTGGTCTTGGTACGACTACTGGGGCCAGGGCACCCTCGTGACCGTGTCA TCT GCTAGC W98YGAGGTGCAATTGGTGGAAAGCGGAGGCGGCCTCGTGAAGCCTGGCGGATCT 211 ntCTGAGACTGAGCTGTGCCGCCAGCGGCTTCACCTTCAGCAACGCCTGGATGAGCTGGGTGCGCCAGGCCCCTGGAAAAGGACTCGAGTGGGTGGGACGGATCAAGAGCAAGACCGATGGCGGCACCACCGACTATGCCGCCCCTGTGAAGGGCCGGTTCACCATCAGCAGGGACGACAGCAAGAACACCCTGTACCTGCAGATGAACAGCCTGAAAACCGAGGACACCGCCGTGTACTACTGCACCACCCCCTGGGAGTACTCTTGGTACGACTACTGGGGCCAGGGCACCCTCGTGACCGTGTCA TCT GCTAGC 90D7CAGGTGCAATTGGTTCAATCTGGTGCTGAAGTAAAAAAACCGGGCGCTTCC 212 ntGTTAAAGTGAGCTGCAAAGCATCCGGATACACCTTCACTTCCTATTACATGCACTGGGTTCGTCAAGCCCCGGGCCAGGGTCTGGAATGGATGGGCATCATTAACCCAAGCGGTGGCTCTACCTCCTACGCGCAGAAATTCCAGGGTCGCGTCACGATGACCCGTGACACTAGCACCTCTACCGTTTATATGGAGCTGTCCAGCCTGCGTTCTGAAGATACTGCAGTGTACTACTGTGCACGCAACTACACTATCGTTGTTTCTCCGTTCGACTATTGGGGTCAAGGCACCCTCGTAACGGTTTCT TCTGCTAGC 90C1CAGGTGCAATTGGTTCAATCTGGTGCTGAAGTAAAAAAACCGGGCGCTTCC 213 ntGTTAAAGTGAGCTGCAAAGCATCCGGATACACCTTCACTTCCTATTACATGCACTGGGTTCGTCAAGCCCCGGGCCAGGGTCTGGAATGGATGGGCATCATTAACCCAAGCGGTGGCTCTACCTCCTACGCGCAGAAATTCCAGGGTCGCGTCACGATGACCCGTGACACTAGCACCTCTACCGTTTATATGGAGCTGTCCAGCCTGCGTTCTGAAGATACTGCAGTGTACTACTGTGCACGCAACTACTTCATCGGTTCTGTTGCTATGGACTATTGGGGTCAAGGCACCCTCGTAACGGTTTCT TCTGCTAGC 5E8 VHCAGGTGCAATTGGTTCAATCTGGTGCTGAAGTAAAAAAACCGGGCGCTTCC 214 ntGTTAAAGTGAGCTGCAAAGCATCCGGATACACCTTCACTTCCTATTACATGCACTGGGTTCGTCAAGCCCCGGGCCAGGGTCTGGAATGGATGGGCATCATTAACCCAAGCGGTGGCTCTACCTCCTACGCGCAGAAATTCCAGGGTCGCGTCACGATGACCCGTGACACTAGCACCTCTACCGTTTATATGGAGCTGTCCAGCCTGCGTTCTGAAGATACTGCAGTGTACTACTGTGCACGCGGTCTGACTTACTCTATGGACTATTGGGGTCAAGGCACCCTCGTAACGGTTTCTTCTGCTAGC 5E8 VLGATATTGTTATGACTCAATCTCCACTGTCTCTGCCGGTGACTCCAGGCGAA 215 ntCCGGCGAGCATTTCTTGCCGTTCCAGCCAGTCTCTGCTGCACTCCAACGGCTACAACTATCTCGATTGGTACCTGCAAAAACCGGGTCAGAGCCCTCAGCTGCTGATCTACCTGGGCTCTAACCGCGCTTCCGGTGTACCGGACCGTTTCAGCGGCTCTGGATCCGGCACCGATTTCACGTTGAAAATCAGCCGTGTTGAAGCAGAAGACGTGGGCGTTTATTACTGTATGCAGGCACTGCAGATTCCAAACACTTTTGGTCAAGGCACCAAGGTCGAAATTAAACGTACG 12A4 VHGAGGTGCAATTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCC 216 ntCTGAGACTCTCCTGTGCAGCCTCCGGATTCACCTTTAGCAGTTATGCCATGAGCTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTCTCAGCTATTAGTGGTAGTGGTGGTAGCACATACTACGCAGACTCCGTGAAGGGCCGGTTCACCATCTCCAGAGACAATCCAAGAACACGCTGTATCTGCAGATGAACAGCCTGAGAGCCGAGGACACGGCCGTATATTACTGTGCGAAATACGCTTACGCTCTGGACTACTGGGGCCAAGGAACCCTGGTCACCGTCTCGAGTGCTAGC 12A4 VLGAAATCGTGTTAACGCAGTCTCCAGGCACCCTGTCTTTGTCTCCAGGGGAA 217 ntAGAGCCACCCTCTCTTGCAGGGCCAGTCAGAGTGTTAGCAGCAGCTACTTAGCCTGGTACCAGCAGAAACCTGGCCAGGCTCCCAGGCTCCTCATCTATGGAGCATCCAGCAGGGCCACTGGCATCCCAGACAGGTTCAGTGGCAGTGGATCCGGGACAGACTTCACTCTCACCATCAGCAGACTGGAGCCTGAAGATTTTGCAGTGTATTACTGTCAGCAGCATGGCAGCAGCAGCACGTTCGGCCAGGGGACCAAAGTGGAAATCAAACGTACG 7A3 VHCAGGTGCAATTGGTTCAATCTGGTGCTGAAGTAAAAAAACCGGGCGCTTCC 218 ntGTTAAAGTGAGCTGCAAAGCATCCGGATACACCTTCACTTCCTATTACATGCACTGGGTTCGTCAAGCCCCGGGCCAGGGTCTGGAATGGATGGGCATCATTAACCCAAGCGGTGGCTCTACCTCCTACGCGCAGAAATTCCAGGGTCGCGTCACGATGACCCGTGACACTAGCACCTCTACCGTTTATATGGAGCTGTCCAGCCTGCGTTCTGAAGATACTGCAGTGTACTACTGTGCACGCGGTGACTTCTCTGCTGGTCGTCTGATGGACTATTGGGGTCAAGGCACCCTCGTAACGGTTTCT TCTGCTAGC 7A3 VLGATATTGTTATGACTCAATCTCCACTGTCTCTGCCGGTGACTCCAGGCGAA 219 ntCCGGCGAGCATTTCTTGCCGTTCCAGCCAGTCTCTGCTGCACTCCAACGGCTACAACTATCTCGATTGGTACCTGCAAAAACCGGGTCAGAGCCCTCAGCTGCTGATCTACCTGGGCTCTAACCGCGCTTCCGGTGTACCGGACCGTTTCAGCGGCTCTGGATCCGGCACCGATTTCACGTTGAAAATCAGCCGTGTTGAAGCAGAAGACGTGGGCGTTTATTACTGTATGCAGGCACTGCAGACCCCACCAATTACCTTTGGTCAAGGCACCAAGGTCGAAATTAAACGTACG 6E10 VHCAGGTGCAATTGGTTCAATCTGGTGCTGAAGTAAAAAAACCGGGCGCTTCC 220 ntGTTAAAGTGAGCTGCAAAGCATCCGGATACACCTTCACTTCCTATTACATGCACTGGGTTCGTCAAGCCCCGGGCCAGGGTCTGGAATGGATGGGCATCATTAACCCAAGCGGTGGCTCTACCTCCTACGCGCAGAAATTCCAGGGTCGCGTCACGATGACCCGTGACACTAGCACCTCTACCGTTTATATGGAGCTGTCCAGCCTGCGTTCTGAAGATACTGCAGTGTACTACTGTGCACGCGGTGACTACAACGCTTTCGACTATTGGGGTCACGGCACCCTCGTAACGGTTTCTTCTGCTAGC 6E10 VLGATATTGTTATGACTCAATCTCCACTGTCTCTGCCGGTGACTCCAGGCGAA 221 ntCCGGCGAGCATTTCTTGCCGTTCCAGCCAGTCTCTGCTGCACTCCAACGGCTACAACTATCTCGATTGGTACCTGCAAAAACCGGGTCAGAGCCCTCAGCTGCTGATCTACCTGGGCTCTAACCGCGCTTCCGGTGTACCGGACCGTTTCAGCGGCTCTGGATCCGGCACCGATTTCACGTTGAAAATCAGCCGTGTTGAAGCAGAAGACGTGGGCGTTTATTACTGTATGCAGGCATGGCATAGCCCAACTTTTGGTCAAGGCACCAAGGTCGAAATTAAACGTACG 12F9 VHCAGGTGCAATTGGTTCAATCTGGTGCTGAAGTAAAAAAACCGGGCGCTTCC 222 ntGTTAAAGTGAGCTGCAAAGCATCCGGATACACCTTCACTTCCTATTACATGCACTGGGTTCGTCAAGCCCCGGGCCAGGGTCTGGAATGGATGGGCATCATTAACCCAAGCGGTGGCTCTACCTCCTACGCGCAGAAATTCCAGGGTCGCGTCACGATGACCCGTGACACTAGCACCTCTACCGTTTATATGGAGCTGTCCAGCCTGCGTTCTGAAGATACTGCAGTGTACTACTGTGCACGCGGTGCTACTTACACTATGGACTATTGGGGTCAAGGCACCCTCGTAACGGTTTCTTCTGCTAGC 12F9 VLGATATTGTTATGACTCAATCTCCACTGTCTCTGCCGGTGACTCCAGGCGAA 223 ntCCGGCGAGCATTTCTTGCCGTTCCAGCCAGTCTCTGCTGCACTCCAACGGCTACAACTATCTCGATTGGTACCTGCAAAAACCGGGTCAGAGCCCTCAGCTGCTGATCTACCTGGGCTCTAACCGCGCTTCCGGTGTACCGGACCGTTTCAGCGGCTCTGGATCCGGCACCGATTTCACGTTGAAAATCAGCCGTGTTGAAGCAGAAGACGTGGGCGTTTATTACTGTATGCAGGCACTGCAGACCCCAATTACTTTTGGTCAAGGCACCAAGGTCGAAATTAAACGTACG pETR11646CAGGTGCAGCTGCAGCAGTCTGGCGCCGAGCTCGTGAAACCTGGCGCCTCC 224 Mov19 VH-GTGAAGATCAGCTGCAAGGCCAGCGGCTACAGCTTCACCGGCTACTTCATG CH1-FcholeAACTGGGTCAAGCAGAGCCACGGCAAGAGCCTGGAATGGATCGGCAGAATC PG/LALACACCCCTACGACGGCGACACCTTCTACAACCAGAACTTCAAGGACAAGGCCACCCTGACCGTGGACAAGAGCAGCAACACCGCCCACATGGAACTGCTGAGCCTGACCAGCGAGGACTTCGCCGTGTACTACTGCACCAGATACGACGGCAGCCGGGCCATGGATTATTGGGGCCAGGGCACCACCGTGACAGTGTCCAGCGCTAGCACCAAGGGCCCCTCCGTGTTCCCCCTGGCCCCCAGCAGCAAGAGCACCAGCGGCGGCACAGCCGCTCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAGCCCGTGACCGTGTCCTGGAACAGCGGAGCCCTGACCTCCGGCGTGCACACCTTCCCCGCCGTGCTGCAGAGTTCTGGCCTGTATAGCCTGAGCAGCGTGGTCACCGTGCCTTCTAGCAGCCTGGGCACCCAGACCTACATCTGCAACGTGAACCACAAGCCCAGCAACACCAAGGTGGACAAGAAGGTGGAGCCCAAGAGCTGCGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAAGCTGCAGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCGGCGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTGCACCCTGCCCCCATCCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTCTCGTGCGCAGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCGTGAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTC TCCCTGTCTCCGGGTAAApETR11647 CAGGTGCAGCTGCAGCAGTCTGGCGCCGAGCTCGTGAAACCTGGCGCCTCC 225Mov19 VH- GTGAAGATCAGCTGCAAGGCCAGCGGCTACAGCTTCACCGGCTACTTCATG CH1-CD3AACTGGGTCAAGCAGAGCCACGGCAAGAGCCTGGAATGGATCGGCAGAATC VH-CL-CACCCCTACGACGGCGACACCTTCTACAACCAGAACTTCAAGGACAAGGCC FcknobACCCTGACCGTGGACAAGAGCAGCAACACCGCCCACATGGAACTGCTGAGC PG/LALACTGACCAGCGAGGACTTCGCCGTGTACTACTGCACCAGATACGACGGCAGCCGGGCCATGGATTATTGGGGCCAGGGCACCACCGTGACAGTGTCCAGCGCTAGCACAAAGGGCCCCAGCGTGTTCCCTCTGGCCCCTAGCAGCAAGAGCACATCTGGCGGAACAGCCGCCCTGGGCTGCCTCGTGAAGGACTACTTTCCCGAGCCTGTGACCGTGTCCTGGAACTCTGGCGCCCTGACAAGCGGCGTGCACACCTTTCCAGCCGTGCTGCAGAGCAGCGGCCTGTACTCTCTGAGCAGCGTGGTCACCGTGCCTAGCAGCAGCCTGGGCACCCAGACCTACATCTGCAACGTGAACCACAAGCCCAGCAACACCAAAGTGGACAAGAAGGTGGAGCCCAAGAGCTGTGATGGCGGAGGAGGGTCCGGAGGCGGAGGATCCGAAGTGCAGCTGGTGGAAAGCGGCGGAGGCCTGGTGCAGCCTAAGGGCTCTCTGAAGCTGAGCTGTGCCGCCAGCGGCTTCACCTTCAACACCTACGCCATGAACTGGGTGCGCCAGGCCCCTGGCAAAGGCCTGGAATGGGTGGCCCGGATCAGAAGCAAGTACAACAATTACGCCACCTACTACGCCGACAGCGTGAAGGACCGGTTCACCATCAGCCGGGACGACAGCCAGAGCATCCTGTACCTGCAGATGAACAACCTGAAAACCGAGGACACCGCCATGTACTACTGCGTGCGGCACGGCAACTTCGGCAACAGCTATGTGTCTTGGTTTGCCTACTGGGGCCAGGGCACCCTCGTGACAGTGTCTGCTGCTAGCGTGGCCGCTCCCTCCGTGTTTATCTTTCCCCCATCCGATGAACAGCTGAAAAGCGGCACCGCCTCCGTCGTGTGTCTGCTGAACAATTTTTACCCTAGGGAAGCTAAAGTGCAGTGGAAAGTGGATAACGCACTGCAGTCCGGCAACTCCCAGGAATCTGTGACAGAACAGGACTCCAAGGACAGCACCTACTCCCTGTCCTCCACCCTGACACTGTCTAAGGCTGATTATGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGTGACAAGACCCACACCTGTCCCCCTTGTCCTGCCCCTGAAGCTGCTGGCGGCCCTTCTGTGTTCCTGTTCCCCCCAAAGCCCAAGGACACCCTGATGATCAGCCGGACCCCCGAAGTGACCTGCGTGGTGGTGGATGTGTCCCACGAGGACCCTGAAGTGAAGTTCAATTGGTACGTGGACGGCGTGGAAGTGCACAACGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCGGCGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATGCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTGTGGTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAA pETR11644GACATCGAGCTGACCCAGAGCCCTGCCTCTCTGGCCGTGTCTCTGGGACAG 226 Mov19 LCAGAGCCATCATCAGCTGCAAGGCCAGCCAGAGCGTGTCCTTTGCCGGCACCTCTCTGATGCACTGGTATCACCAGAAGCCCGGCCAGCAGCCCAAGCTGCTGATCTACAGAGCCAGCAACCTGGAAGCCGGCGTGCCCACAAGATTTTCCGGCAGCGGCAGCAAGACCGACTTCACCCTGAACATCCACCCCGTGGAAGAAGAGGACGCCGCCACCTACTACTGCCAGCAGAGCAGAGAGTACCCCTACACCTTCGGCGGAGGCACCAAGCTGGAAATCAAGCGTACGGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGT Seq Variant Sequence ID No16D5 GAGGTGCAATTGGTTGAATCTGGTGGTGGTCTGGTAAAACCGGGCGGTTCCC 261 VH_D52dETGCGTCTGAGCTGCGCGGCTTCCGGATTCACCTTCTCCAACGCGTGGATGAGCTGGGTTCGCCAGGCCCCGGGCAAAGGCCTCGAGTGGGTTGGTCGTATCAAGTCTAAAACTGAGGGTGGCACCACGGATTACGCGGCTCCAGTTAAAGGTCGTTTTACCATTTCCCGCGACGATAGCAAAAACACTCTGTATCTGCAGATGAACTCTCTGAAAACTGAAGACACCGCAGTCTACTACTGTACTACCCCGTGGGAATGGTCTTGGTACGATTATTGGGGCCAGGGCACGCTGGTTACGGTGTCTTCC 16D5GAGGTGCAATTGGTTGAATCTGGTGGTGGTCTGGTAAAACCGGGCGGTTCCC 262 VH_D52dQTGCGTCTGAGCTGCGCGGCTTCCGGATTCACCTTCTCCAACGCGTGGATGAGCTGGGTTCGCCAGGCCCCGGGCAAAGGCCTCGAGTGGGTTGGTCGTATCAAGTCTAAAACTCAGGGTGGCACCACGGATTACGCGGCTCCAGTTAAAGGTCGTTTTACCATTTCCCGCGACGATAGCAAAAACACTCTGTATCTGCAGATGAACTCTCTGAAAACTGAAGACACCGCAGTCTACTACTGTACTACCCCGTGGGAATGGTCTTGGTACGATTATTGGGGCCAGGGCACGCTGGTTACGGTGTCTTCC CD3_VHGAGGTGCAGCTGCTGGAATCTGGCGGCGGACTGGTGCAGCCTGGCGGATCTC 263 N100ATGAGACTGAGCTGTGCCGCCAGCGGCTTCACCTTCAGCACCTACGCCATGAACTGGGTGCGCCAGGCCCCTGGCAAAGGCCTGGAATGGGTGTCCCGGATCAGAAGCAAGTACAACAACTACGCCACCTACTACGCCGACAGCGTGAAGGGCCGGTTCACCATCAGCCGGGACGACAGCAAGAACACCCTGTACCTGCAGATGAACAGCCTGCGGGCCGAGGACACCGCCGTGTACTATTGTGTGCGGCACGGCAACTTCGGCGCCAGCTATGTGTCTTGGTTTGCCTACTGGGGCCAGGGCACCCTCGTGA CCGTGTCAAGC CD3_VHGAGGTGCAGCTGCTGGAATCTGGCGGCGGACTGGTGCAGCCTGGCGGATCTC 264 S100aATGAGACTGAGCTGTGCCGCCAGCGGCTTCACCTTCAGCACCTACGCCATGAACTGGGTGCGCCAGGCCCCTGGCAAAGGCCTGGAATGGGTGTCCCGGATCAGAAGCAAGTACAACAACTACGCCACCTACTACGCCGACAGCGTGAAGGGCCGGTTCACCATCAGCCGGGACGACAGCAAGAACACCCTGTACCTGCAGATGAACAGCCTGCGGGCCGAGGACACCGCCGTGTACTATTGTGTGCGGCACGGCAACTTCGGCAACGCCTATGTGTCTTGGTTTGCCTACTGGGGCCAGGGCACCCTCGTGA CCGTGTCAAGC 16D5GAGGTGCAATTGGTTGAATCTGGTGGTGGTCTGGTAAAACCGGGCGGTTCCC 265 [VHCH1]-TGCGTCTGAGCTGCGCGGCTTCCGGATTCACCTTCTCCAACGCGTGGATGAG CD3[VHCCTGGGTTCGCCAGGCCCCGGGCAAAGGCCTCGAGTGGGTTGGTCGTATCAAG H1-TCTAAAACTGACGGTGGCACCACGGATTACGCGGCTCCAGTTAAAGGTCGTT N100A]-TTACCATTTCCCGCGACGATAGCAAAAACACTCTGTATCTGCAGATGAACTC Fcknob_TCTGAAAACTGAAGACACCGCAGTCTACTACTGTACTACCCCGTGGGAATGG PGLALATCTTGGTACGATTATTGGGGCCAGGGCACGCTGGTTACGGTGTCTTCCGCTAGCACAAAGGGCCCTAGCGTGTTCCCTCTGGCCCCCAGCAGCAAGAGCACAAGCGGCGGAACAGCCGCCCTGGGCTGCCTCGTGAAGGACTACTTCCCCGAGCCCGTGACAGTGTCTTGGAACAGCGGAGCCCTGACAAGCGGCGTGCACACTTTCCCTGCCGTGCTGCAGAGCAGCGGCCTGTACTCCCTGAGCAGCGTGGTCACCGTGCCTAGCAGCAGCCTGGGCACCCAGACCTACATCTGCAACGTGAACCACAAGCCCAGCAACACCAAAGTGGACAAGAAGGTGGAGCCCAAGAGCTGTGATGGCGGAGGAGGGTCCGGAGGCGGAGGATCCGAGGTGCAGCTGCTGGAATCTGGCGGCGGACTGGTGCAGCCTGGCGGATCTCTGAGACTGAGCTGTGCCGCCAGCGGCTTCACCTTCAGCACCTACGCCATGAACTGGGTGCGCCAGGCCCCTGGCAAAGGCCTGGAATGGGTGTCCCGGATCAGAAGCAAGTACAACAACTACGCCACCTACTACGCCGACAGCGTGAAGGGCCGGTTCACCATCAGCCGGGACGACAGCAAGAACACCCTGTACCTGCAGATGAACAGCCTGCGGGCCGAGGACACCGCCGTGTACTATTGTGTGCGGCACGGCAACTTCGGCGCCAGCTATGTGTCTTGGTTTGCCTACTGGGGCCAGGGCACCCTCGTGACCGTGTCAAGCGCTAGTACCAAGGGCCCCAGCGTGTTCCCCCTGGCACCCAGCAGCAAGAGCACATCTGGCGGAACAGCCGCTCTGGGCTGTCTGGTGAAAGACTACTTCCCCGAGCCCGTGACCGTGTCTTGGAACTCTGGCGCCCTGACCAGCGGCGTGCACACCTTTCCAGCCGTGCTGCAGAGCAGCGGCCTGTACTCCCTGTCCTCCGTGGTCACCGTGCCCTCTAGCTCCCTGGGAACACAGACATATATCTGTAATGTCAATCACAAGCCTTCCAACACCAAAGTCGATAAGAAAGTCGAGCCCAAGAGCTGCGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAAGCTGCAGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCGGCGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATGCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTGTGGTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAA 16D5-GAGGTGCAATTGGTTGAATCTGGTGGTGGTCTGGTAAAACCGGGCGGTTCCC 266 Fchole-TGCGTCTGAGCTGCGCGGCTTCCGGATTCACCTTCTCCAACGCGTGGATGAG PGLALACTGGGTTCGCCAGGCCCCGGGCAAAGGCCTCGAGTGGGTTGGTCGTATCAAGTCTAAAACTGACGGTGGCACCACGGATTACGCGGCTCCAGTTAAAGGTCGTTTTACCATTTCCCGCGACGATAGCAAAAACACTCTGTATCTGCAGATGAACTCTCTGAAAACTGAAGACACCGCAGTCTACTACTGTACTACCCCGTGGGAATGGTCTTGGTACGATTATTGGGGCCAGGGCACGCTGGTTACGGTGTCTTCCGCTAGCACCAAGGGCCCCTCCGTGTTCCCCCTGGCCCCCAGCAGCAAGAGCACCAGCGGCGGCACAGCCGCTCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAGCCCGTGACCGTGTCCTGGAACAGCGGAGCCCTGACCTCCGGCGTGCACACCTTCCCCGCCGTGCTGCAGAGTTCTGGCCTGTATAGCCTGAGCAGCGTGGTCACCGTGCCTTCTAGCAGCCTGGGCACCCAGACCTACATCTGCAACGTGAACCACAAGCCCAGCAACACCAAGGTGGACAAGAAGGTGGAGCCCAAGAGCTGCGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAAGCTGCAGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCGGCGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTGCACCCTGCCCCCATCCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTCTCGTGCGCAGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCGTGAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCGCTTCACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAA CD3-CLCCAGGCCGTCGTGACCCAGGAACCCAGCCTGACAGTGTCTCCTGGCGGCACCG 267TGACCCTGACATGTGGCAGTTCTACAGGCGCCGTGACCACCAGCAACTACGCCAACTGGGTGCAGGAAAAGCCCGGCCAGGCCTTCAGAGGACTGATCGGCGGCACCAACAAGAGAGCCCCTGGCACCCCTGCCAGATTCAGCGGATCTCTGCTGGGAGGAAAGGCCGCCCTGACACTGTCTGGCGCCCAGCCAGAAGATGAGGCCGAGTACTACTGCGCCCTGTGGTACAGCAACCTGTGGGTGTTCGGCGGAGGCACCAAGCTGACAGTCCTAGGTCAACCCAAGGCTGCCCCCAGCGTGACCCTGTTCCCCCCCAGCAGCGAGGAACTGCAGGCCAACAAGGCCACCCTGGTCTGCCTGATCAGCGACTTCTACCCAGGCGCCGTGACCGTGGCCTGGAAGGCCGACAGCAGCCCCGTGAAGGCCGGCGTGGAGACCACCACCCCCAGCAAGCAGAGCAACAACAAGTACGCCGCCAGCAGCTACCTGAGCCTGACCCCCGAGCAGTGGAAGAGCCACAGGTCCTACAGCTGCCAGGTGACCCACGAGGGCAGCACCGTGGAGAAAACCGTGGCCCCCACCGAGTGCAGC 16D5GAGGTGCAATTGGTTGAATCTGGTGGTGGTCTGGTAAAACCGGGCGGTTCCC 268 [VHCH1]-TGCGTCTGAGCTGCGCGGCTTCCGGATTCACCTTCTCCAACGCGTGGATGAG CD3[VHCH1-CTGGGTTCGCCAGGCCCCGGGCAAAGGCCTCGAGTGGGTTGGTCGTATCAAG S100aA]-TCTAAAACTGACGGTGGCACCACGGATTACGCGGCTCCAGTTAAAGGTCGTT Fcknob_TTACCATTTCCCGCGACGATAGCAAAAACACTCTGTATCTGCAGATGAACTC PGLALA TCTGAAAACTGAAGACACCGCAGTCTACTACTGTACTACCCCGTGGGAATGGTCTTGGTACGATTATTGGGGCCAGGGCACGCTGGTTACGGTGTCTTCCGCTAGCACAAAGGGCCCTAGCGTGTTCCCTCTGGCCCCCAGCAGCAAGAGCACAAGCGGCGGAACAGCCGCCCTGGGCTGCCTCGTGAAGGACTACTTCCCCGAGCCCGTGACAGTGTCTTGGAACAGCGGAGCCCTGACAAGCGGCGTGCACACTTTCCCTGCCGTGCTGCAGAGCAGCGGCCTGTACTCCCTGAGCAGCGTGGTCACCGTGCCTAGCAGCAGCCTGGGCACCCAGACCTACATCTGCAACGTGAACCACAAGCCCAGCAACACCAAAGTGGACAAGAAGGTGGAGCCCAAGAGCTGTGATGGCGGAGGAGGGTCCGGAGGCGGAGGATCCGAGGTGCAGCTGCTGGAATCTGGCGGCGGACTGGTGCAGCCTGGCGGATCTCTGAGACTGAGCTGTGCCGCCAGCGGCTTCACCTTCAGCACCTACGCCATGAACTGGGTGCGCCAGGCCCCTGGCAAAGGCCTGGAATGGGTGTCCCGGATCAGAAGCAAGTACAACAACTACGCCACCTACTACGCCGACAGCGTGAAGGGCCGGTTCACCATCAGCCGGGACGACAGCAAGAACACCCTGTACCTGCAGATGAACAGCCTGCGGGCCGAGGACACCGCCGTGTACTATTGTGTGCGGCACGGCAACTTCGGCAACGCCTATGTGTCTTGGTTTGCCTACTGGGGCCAGGGCACCCTCGTGACCGTGTCAAGCGCTAGTACCAAGGGCCCCAGCGTGTTCCCCCTGGCACCCAGCAGCAAGAGCACATCTGGCGGAACAGCCGCTCTGGGCTGTCTGGTGAAAGACTACTTCCCCGAGCCCGTGACCGTGTCTTGGAACTCTGGCGCCCTGACCAGCGGCGTGCACACCTTTCCAGCCGTGCTGCAGAGCAGCGGCCTGTACTCCCTGTCCTCCGTGGTCACCGTGCCCTCTAGCTCCCTGGGAACACAGACATATATCTGTAATGTCAATCACAAGCCTTCCAACACCAAAGTCGATAAGAAAGTCGAGCCCAAGAGCTGCGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAAGCTGCAGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCGGCGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATGCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTGTGGTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAA 9D11CAGGTGCAATTGGTTCAATCTGGTGCTGAAGTAAAAAAACCGGGCGCTTCCG 269 [VHCH1]-TTAAAGTGAGCTGCAAAGCATCCGGATACACCTTCACTTCCTATTACATGCA CD3[VHCL-CTGGGTTCGTCAAGCCCCGGGCCAGGGTCTGGAATGGATGGGCATCATTAAC N100A]-CCAAGCGGTGGCCCTACCTCCTACGCGCAGAAATTCCAGGGTCGCGTCACGA Fcknob_TGACCCGTGACACTAGCACCTCTACCGTTTATATGGAGCTGTCCAGCCTGCG PGLALATTCTGAAGATACTGCAGTGTACTACTGTGCACGCGGTGACTTCGCTTGGCTGGACTATTGGGGTCAAGGCACCCTCGTAACGGTTTCTTCTGCTAGCACAAAGGGCCCCAGCGTGTTCCCTCTGGCCCCTAGCAGCAAGAGCACATCTGGCGGAACAGCCGCCCTGGGCTGCCTCGTGAAGGACTACTTTCCCGAGCCTGTGACCGTGTCCTGGAACTCTGGCGCCCTGACAAGCGGCGTGCACACCTTTCCAGCCGTGCTGCAGAGCAGCGGCCTGTACTCTCTGAGCAGCGTGGTCACCGTGCCTAGCAGCAGCCTGGGCACCCAGACCTACATCTGCAACGTGAACCACAAGCCCAGCAACACCAAAGTGGACAAGAAGGTGGAGCCCAAGAGCTGTGATGGCGGAGGAGGGTCCGGAGGCGGAGGATCCGAGGTGCAGCTGCTGGAATCTGGCGGCGGACTGGTGCAGCCTGGCGGATCTCTGAGACTGAGCTGTGCCGCCAGCGGCTTCACCTTCAGCACCTACGCCATGAACTGGGTGCGCCAGGCCCCTGGCAAAGGCCTGGAATGGGTGTCCCGGATCAGAAGCAAGTACAACAACTACGCCACCTACTACGCCGACAGCGTGAAGGGCCGGTTCACCATCAGCCGGGACGACAGCAAGAACACCCTGTACCTGCAGATGAACAGCCTGCGGGCCGAGGACACCGCCGTGTACTATTGTGTGCGGCACGGCAACTTCGGCGCCAGCTATGTGTCTTGGTTTGCCTACTGGGGCCAGGGCACCCTCGTGACCGTGTCAAGCGCTAGTGTGGCCGCTCCCTCCGTGTTTATCTTTCCCCCATCCGATGAACAGCTGAAAAGCGGCACCGCCTCCGTCGTGTGTCTGCTGAACAATTTTTACCCTAGGGAAGCTAAAGTGCAGTGGAAAGTGGATAACGCACTGCAGTCCGGCAACTCCCAGGAATCTGTGACAGAACAGGACTCCAAGGACAGCACCTACTCCCTGTCCTCCACCCTGACACTGTCTAAGGCTGATTATGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGTGACAAGACCCACACCTGTCCCCCTTGTCCTGCCCCTGAAGCTGCTGGCGGCCCTTCTGTGTTCCTGTTCCCCCCAAAGCCCAAGGACACCCTGATGATCAGCCGGACCCCCGAAGTGACCTGCGTGGTGGTGGATGTGTCCCACGAGGACCCTGAAGTGAAGTTCAATTGGTACGTGGACGGCGTGGAAGTGCACAACGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCGGCGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATGCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTGTGGTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAA 9D11-CAGGTGCAATTGGTTCAATCTGGTGCTGAAGTAAAAAAACCGGGCGCTTCCG 270 FcholeTTAAAGTGAGCTGCAAAGCATCCGGATACACCTTCACTTCCTATTACATGCACTGGGTTCGTCAAGCCCCGGGCCAGGGTCTGGAATGGATGGGCATCATTAACCCAAGCGGTGGCCCTACCTCCTACGCGCAGAAATTCCAGGGTCGCGTCACGATGACCCGTGACACTAGCACCTCTACCGTTTATATGGAGCTGTCCAGCCTGCGTTCTGAAGATACTGCAGTGTACTACTGTGCACGCGGTGACTTCGCTTGGCTGGACTATTGGGGTCAAGGCACCCTCGTAACGGTTTCTTCTGCTAGCACCAAGGGCCCCTCCGTGTTCCCCCTGGCCCCCAGCAGCAAGAGCACCAGCGGCGGCACAGCCGCTCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAGCCCGTGACCGTGTCCTGGAACAGCGGAGCCCTGACCTCCGGCGTGCACACCTTCCCCGCCGTGCTGCAGAGTTCTGGCCTGTATAGCCTGAGCAGCGTGGTCACCGTGCCTTCTAGCAGCCTGGGCACCCAGACCTACATCTGCAACGTGAACCACAAGCCCAGCAACACCAAGGTGGACAAGAAGGTGGAGCCCAAGAGCTGCGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAAGCTGCAGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCGGCGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTGCACCCTGCCCCCATCCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTCTCGTGCGCAGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCGTGAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAA 9D11_LCGATATTGTTATGACTCAATCTCCACTGTCTCTGCCGGTGACTCCAGGCGAAC 271 [N95Q]CGGCGAGCATTTCTTGCCGTTCCAGCCAGTCTCTGCTGCACTCCAACGGCTACAACTATCTCGATTGGTACCTGCAAAAACCGGGTCAGAGCCCTCAGCTGCGATCTACCTGGGCTCTAACCGCGCTTCCGGTGTACCGGACCGTTTCAGCGGCTCTGGATCCGGCACCGATTTCACGTTGAAAATCAGCCGTGTTGAAGCAGAAGACGTGGGCGTTTATTACTGTATGCAGGCAAGCATTATGCAGCGGACTTTTGGTCAAGGCACCAAGGTCGAAATTAAACGTACGGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGT CD3_VLCH1CAGGCCGTCGTGACCCAGGAACCCAGCCTGACAGTGTCTCCTGGCGGCACCG 272TGACCCTGACATGTGGCAGTTCTACAGGCGCCGTGACCACCAGCAACTACGCCAACTGGGTGCAGGAAAAGCCCGGCCAGGCCTTCAGAGGACTGATCGGCGGCACCAACAAGAGAGCCCCTGGCACCCCTGCCAGATTCAGCGGATCTCTGCTGGGAGGAAAGGCCGCCCTGACACTGTCTGGCGCCCAGCCAGAAGATGAGGCCGAGTACTACTGCGCCCTGTGGTACAGCAACCTGTGGGTGTTCGGCGGAGGCACCAAGCTGACAGTGCTGAGCAGCGCTTCCACCAAAGGCCCTTCCGTGTTTCCTCTGGCTCCTAGCTCCAAGTCCACCTCTGGAGGCACCGCTGCTCTCGGATGCCTCGTGAAGGATTATTTTCCTGAGCCTGTGACAGTGTCCTGGAATAGCGGAGCACTGACCTCTGGAGTGCATACTTTCCCCGCTGTGCTGCAGTCCTCTGGACTGTACAGCCTGAGCAGCGTGGTGACAGTGCCCAGCAGCAGCCTGGGCACCCAGACCTACATCTGCAACGTGAACCACAAGCCCAGCAACACCAAGGTGGACAAGAAG GTGGAACCCAAGTCTTGT9D11 CAGGTGCAATTGGTTCAATCTGGTGCTGAAGTAAAAAAACCGGGCGCTTCCG 273 [VHCH1]-TTAAAGTGAGCTGCAAAGCATCCGGATACACCTTCACTTCCTATTACATGCA CD3[VHCH1-CTGGGTTCGTCAAGCCCCGGGCCAGGGTCTGGAATGGATGGGCATCATTAAC S100aA]-CCAAGCGGTGGCCCTACCTCCTACGCGCAGAAATTCCAGGGTCGCGTCACGA Fcknob_TGACCCGTGACACTAGCACCTCTACCGTTTATATGGAGCTGTCCAGCCTGCG PGLALATTCTGAAGATACTGCAGTGTACTACTGTGCACGCGGTGACTTCGCTTGGCTGGACTATTGGGGTCAAGGCACCCTCGTAACGGTTTCTTCTGCTAGCACAAAGGGCCCCAGCGTGTTCCCTCTGGCCCCTAGCAGCAAGAGCACATCTGGCGGAACAGCCGCCCTGGGCTGCCTCGTGAAGGACTACTTTCCCGAGCCTGTGACCGTGTCCTGGAACTCTGGCGCCCTGACAAGCGGCGTGCACACCTTTCCAGCCGTGCTGCAGAGCAGCGGCCTGTACTCTCTGAGCAGCGTGGTCACCGTGCCTAGCAGCAGCCTGGGCACCCAGACCTACATCTGCAACGTGAACCACAAGCCCAGCAACACCAAAGTGGACAAGAAGGTGGAGCCCAAGAGCTGTGATGGCGGAGGAGGGTCCGGAGGCGGAGGATCCGAGGTGCAGCTGCTGGAATCTGGCGGCGGACTGGTGCAGCCTGGCGGATCTCTGAGACTGAGCTGTGCCGCCAGCGGCTTCACCTTCAGCACCTACGCCATGAACTGGGTGCGCCAGGCCCCTGGCAAAGGCCTGGAATGGGTGTCCCGGATCAGAAGCAAGTACAACAACTACGCCACCTACTACGCCGACAGCGTGAAGGGCCGGTTCACCATCAGCCGGGACGACAGCAAGAACACCCTGTACCTGCAGATGAACAGCCTGCGGGCCGAGGACACCGCCGTGTACTATTGTGTGCGGCACGGCAACTTCGGCAACGCCTATGTGTCTTGGTTTGCCTACTGGGGCCAGGGCACCCTCGTGACCGTGTCAAGCGCTAGTGTGGCCGCTCCCTCCGTGTTTATCTTTCCCCCATCCGATGAACAGCTGAAAAGCGGCACCGCCTCCGTCGTGTGTCTGCTGAACAATTTTTACCCTAGGGAAGCTAAAGTGCAGTGGAAAGTGGATAACGCACTGCAGTCCGGCAACTCCCAGGAATCTGTGACAGAACAGGACTCCAAGGACAGCACCTACTCCCTGTCCTCCACCCTGACACTGTCTAAGGCTGATTATGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGTGACAAGACCCACACCTGTCCCCCTTGTCCTGCCCCTGAAGCTGCTGGCGGCCCTTCTGTGTTCCTGTTCCCCCCAAAGCCCAAGGACACCCTGATGATCAGCCGGACCCCCGAAGTGACCTGCGTGGTGGTGGATGTGTCCCACGAGGACCCTGAAGTGAAGTTCAATTGGTACGTGGACGGCGTGGAAGTGCACAACGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCGGCGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATGCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTGTGGTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAA Seq Name Sequence ID No 16D5GAGGTGCAATTGGTGGAAAGCGGAGGCGGCCTCGTGAAGCCTGGCGGATCTCTG 287 variantAGACTGAGCTGTGCCGCCAGCGGCTTCACCTTCAGCAACGCCTGGATGAGCTGG W96Y/D52EGTGCGCCAGGCCCCTGGAAAAGGACTCGAGTGGGTGGGACGGATCAAGAGCAAG VHACCGAGGGCGGCACCACCGACTATGCCGCCCCTGTGAAGGGCCGGTTCACCATCAGCAGGGACGACAGCAAGAACACCCTGTACCTGCAGATGAACAGCCTGAAAACCGAGGACACCGCCGTGTACTACTGCACCACCCCCTACGAGTGGTCTTGGTACGACTACTGGGGCCAGGGCACCCTCGTGACCGTGTCATCT W96Y/D52E_GAGGTGCAATTGGTGGAAAGCGGAGGCGGCCTCGTGAAGCCTGGCGGATCTCTG 288 CD3-AGACTGAGCTGTGCCGCCAGCGGCTTCACCTTCAGCAACGCCTGGATGAGCTGG VHCH1_GTGCGCCAGGCCCCTGGAAAAGGACTCGAGTGGGTGGGACGGATCAAGAGCAAG Fc-knob_ACCGAGGGCGGCACCACCGACTATGCCGCCCCTGTGAAGGGCCGGTTCACCATC PGLALAAGCAGGGACGACAGCAAGAACACCCTGTACCTGCAGATGAACAGCCTGAAAACC pETR14945GAGGACACCGCCGTGTACTACTGCACCACCCCCTACGAGTGGTCTTGGTACGACTACTGGGGCCAGGGCACCCTCGTGACCGTGTCATCTGCTAGCACAAAGGGCCCTAGCGTGTTCCCTCTGGCCCCCAGCAGCAAGAGCACAAGCGGCGGAACAGCCGCCCTGGGCTGCCTCGTGAAGGACTACTTCCCCGAGCCCGTGACAGTGTCTTGGAACAGCGGAGCCCTGACAAGCGGCGTGCACACCTTCCCTGCCGTGCTGCAGAGCAGCGGCCTGTACTCCCTGAGCAGCGTGGTCACCGTGCCTAGCAGCAGCCTGGGCACCCAGACCTACATCTGCAACGTGAACCACAAGCCCAGCAACACCAAAGTGGACAAGAAGGTGGAGCCCAAGAGCTGTGATGGCGGAGGAGGGTCCGGAGGCGGAGGATCCGAGGTGCAGCTGCTGGAATCTGGCGGCGGACTGGTGCAGCCTGGCGGATCTCTGAGACTGAGCTGTGCCGCCAGCGGCTTCACCTTCAGCACCTACGCCATGAACTGGGTGCGCCAGGCCCCTGGCAAAGGCCTGGAATGGGTGTCCCGGATCAGAAGCAAGTACAACAACTACGCCACCTACTACGCCGACAGCGTGAAGGGCCGGTTCACCATCAGCCGGGACGACAGCAAGAACACCCTGTACCTGCAGATGAACAGCCTGCGGGCCGAGGACACCGCCGTGTACTATTGTGTGCGGCACGGCAACTTCGGCAACAGCTATGTGTCTTGGTTTGCCTACTGGGGCCAGGGCACCCTCGTGACCGTGTCAAGCGCTAGTACCAAGGGCCCCAGCGTGTTCCCCCTGGCACCCAGCAGCAAGAGCACATCTGGCGGAACAGCCGCTCTGGGCTGTCTGGTGAAAGACTACTTCCCCGAGCCCGTGACCGTGTCTTGGAACTCTGGCGCCCTGACCAGCGGCGTGCACACCTTTCCAGCCGTGCTGCAGAGCAGCGGCCTGTACTCCCTGTCCTCCGTGGTCACCGTGCCCTCTAGCTCCCTGGGAACACAGACATATATCTGTAATGTCAATCACAAGCCTTCCAACACCAAAGTCGATAAGAAAGTCGAGCCCAAGAGCTGCGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAAGCTGCAGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCGGCGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATGCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTGTGGTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCC CTGTCTCCGGGTAAAW96Y/D52E_ GAGGTGCAATTGGTGGAAAGCGGAGGCGGCCTCGTGAAGCCTGGCGGATCTCTG 289Fc-hole_ AGACTGAGCTGTGCCGCCAGCGGCTTCACCTTCAGCAACGCCTGGATGAGCTGG PGLALA_GTGCGCCAGGCCCCTGGAAAAGGACTCGAGTGGGTGGGACGGATCAAGAGCAAG HYRFACCGAGGGCGGCACCACCGACTATGCCGCCCCTGTGAAGGGCCGGTTCACCATC pETR14946AGCAGGGACGACAGCAAGAACACCCTGTACCTGCAGATGAACAGCCTGAAAACCGAGGACACCGCCGTGTACTACTGCACCACCCCCTACGAGTGGTCTTGGTACGACTACTGGGGCCAGGGCACCCTCGTGACCGTGTCATCTGCTAGCACCAAGGGCCCCTCCGTGTTCCCCCTGGCCCCCAGCAGCAAGAGCACCAGCGGCGGCACAGCCGCTCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAGCCCGTGACCGTGTCCTGGAACAGCGGAGCCCTGACCTCCGGCGTGCACACCTTCCCCGCCGTGCTGCAGAGTTCTGGCCTGTATAGCCTGAGCAGCGTGGTCACCGTGCCTTCTAGCAGCCTGGGCACCCAGACCTACATCTGCAACGTGAACCACAAGCCCAGCAACACCAAGGTGGACAAGAAGGTGGAGCCCAAGAGCTGCGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAAGCTGCAGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCGGCGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTGCACCCTGCCCCCATCCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTCTCGTGCGCAGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCGTGAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCGCTTCACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAA 14B1GAGGTGCAATTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTG 290 VHAGACTCTCCTGTGCAGCCTCCGGATTCACCTTTAGCAGTTATGCCATGAGCTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTCTCAGCTATTAGTGGTAGTGGTGGTAGCACATACTACGCAGACTCCGTGAAGGGCCGGTTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTATCTGCAGATGAACAGCCTGAGAGCCGAGGACACGGCCGTATATTACTGTGCGCGTGGTGACTACCGTTACCGTTACTTCGACTACTGGGGCCAAGGAACCCTGGTCACCGTCTCGAGT 14B1TCTTCTGAACTGACTCAAGATCCAGCTGTTAGCGTGGCTCTGGGTCAGACTGTA 291 VLCGTATCACCTGCCAAGGCGATTCTCTGCGCTCCTACTACGCAAGCTGGTACCAGCAGAAACCGGGTCAGGCCCCAGTTCTGGTGATTTACGGCAAAAACAACCGTCCGTCTGGGATCCCGGACCGTTTCTCCGGCAGCTCTTCCGGTAACACGGCGAGCCTCACCATCACTGGCGCTCAAGCAGAAGACGAGGCCGACTATTACTGTAACTCTCGGGAAAGCCCACCAACCGGCCTGGTTGTCTTCGGTGGCGGTACCAAGCTGACCGTC CTA 14B1[EE]_GAGGTGCAATTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTG 292 CD3[VLCH1]_AGACTCTCCTGTGCAGCCTCCGGATTCACCTTTAGCAGTTATGCCATGAGCTGG Fc-GTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTCTCAGCTATTAGTGGTAGT knob_GGTGGTAGCACATACTACGCAGACTCCGTGAAGGGCCGGTTCACCATCTCCAGA PGLALAGACAATTCCAAGAACACGCTGTATCTGCAGATGAACAGCCTGAGAGCCGAGGAC pETR14976ACGGCCGTATATTACTGTGCGCGTGGTGACTACCGTTACCGTTACTTCGACTACTGGGGCCAAGGAACCCTGGTCACCGTCTCGAGTGCTAGCACCAAGGGCCCCTCCGTGTTTCCTCTGGCCCCTTCCAGCAAGTCCACCTCTGGCGGAACTGCCGCTCTGGGCTGCCTGGTGGAAGATTACTTCCCCGAGCCCGTGACCGTGTCCTGGAATTCTGGCGCTCTGACCTCCGGCGTGCACACCTTTCCAGCTGTGCTGCAGTCCTCCGGCCTGTACTCCCTGTCCTCCGTCGTGACAGTGCCCTCCAGCTCTCTGGGCACCCAGACCTACATCTGCAACGTGAACCACAAGCCCTCCAACACCAAGGTGGACGAGAAGGTGGAACCCAAGTCCTGCGACGGTGGCGGAGGTTCCGGAGGCGGAGGATCCCAGGCTGTCGTGACCCAGGAACCCTCCCTGACAGTGTCTCCTGGCGGCACCGTGACCCTGACCTGTGGATCTTCTACCGGCGCTGTGACCACCTCCAACTACGCCAATTGGGTGCAGGAAAAGCCCGGCCAGGCCTTCAGAGGACTGATCGGCGGCACCAACAAGAGAGCCCCTGGCACCCCTGCCAGATTCTCCGGTTCTCTGCTGGGCGGCAAGGCTGCCCTGACTCTGTCTGGTGCTCAGCCTGAGGACGAGGCCGAGTACTACTGCGCCCTGTGGTACTCCAACCTGTGGGTGTTCGGCGGAGGCACCAAGCTGACCGTGCTGTCCAGCGCTTCCACCAAGGGACCCAGTGTGTTCCCCCTGGCCCCCAGCTCCAAGTCTACATCCGGTGGCACAGCTGCCCTGGGATGTCTCGTGAAGGACTACTTTCCTGAGCCTGTGACAGTGTCTTGGAACAGCGGAGCCCTGACCAGCGGAGTGCACACATTCCCTGCAGTGCTGCAGAGCAGCGGCCTGTATAGCCTGAGCAGCGTCGTGACCGTGCCTTCCTCTAGCCTGGGAACACAGACATATATCTGTAATGTGAATCATAAGCCCAGTAATACCAAAGTGGATAAGAAAGTGGAACCTAAGAGCTGCGATAAGACCCACACCTGTCCCCCCTGCCCTGCTCCTGAAGCTGCTGGTGGCCCTAGCGTGTTCCTGTTCCCCCCAAAGCCCAAGGACACCCTGATGATCTCCCGGACCCCCGAAGTGACCTGCGTGGTGGTGGATGTGTCCCACGAGGACCCTGAAGTGAAGTTCAATTGGTACGTGGACGGCGTGGAAGTGCACAACGCCAAGACCAAGCCTAGAGAGGAACAGTACAACTCCACCTACCGGGTGGTGTCCGTGCTGACAGTGCTGCACCAGGACTGGCTGAACGGCAAAGAGTACAAGTGCAAGGTGTCCAACAAGGCCCTGGGCGCTCCCATCGAAAAGACCATCTCCAAGGCCAAGGGCCAGCCCCGGGAACCCCAGGTGTACACCCTGCCCCCATGCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTGTGGTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAA 14B1[EE]_GAGGTGCAATTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTG 293 Fc-AGACTCTCCTGTGCAGCCTCCGGATTCACCTTTAGCAGTTATGCCATGAGCTGG hole_GTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTCTCAGCTATTAGTGGTAGT PGLALAGGTGGTAGCACATACTACGCAGACTCCGTGAAGGGCCGGTTCACCATCTCCAGA pETR14977GACAATTCCAAGAACACGCTGTATCTGCAGATGAACAGCCTGAGAGCCGAGGACACGGCCGTATATTACTGTGCGCGTGGTGACTACCGTTACCGTTACTTCGACTACTGGGGCCAAGGAACCCTGGTCACCGTCTCGAGTGCTAGCACCAAGGGCCCCTCCGTGTTCCCCCTGGCCCCCAGCAGCAAGAGCACCAGCGGCGGCACAGCCGCTCTGGGCTGCCTGGTCGAGGACTACTTCCCCGAGCCCGTGACCGTGTCCTGGAACAGCGGAGCCCTGACCTCCGGCGTGCACACCTTCCCCGCCGTGCTGCAGAGTTCTGGCCTGTATAGCCTGAGCAGCGTGGTCACCGTGCCTTCTAGCAGCCTGGGCACCCAGACCTACATCTGCAACGTGAACCACAAGCCCAGCAACACCAAGGTGGACGAGAAGGTGGAGCCCAAGAGCTGCGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAAGCTGCAGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCGGCGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTGCACCCTGCCCCCATCCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTCTCGTGCGCAGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCGTGAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAA 14B1 LCTCTTCTGAACTGACTCAAGATCCAGCTGTTAGCGTGGCTCTGGGTCAGACTGTA 294 [KK]CGTATCACCTGCCAAGGCGATTCTCTGCGCTCCTACTACGCAAGCTGGTACCAG ConstantCAGAAACCGGGTCAGGCCCCAGTTCTGGTGATTTACGGCAAAAACAACCGTCCG lambdaTCTGGGATCCCGGACCGTTTCTCCGGCAGCTCTTCCGGTAACACGGCGAGCCTC pETR14979ACCATCACTGGCGCTCAAGCAGAAGACGAGGCCGACTATTACTGTAACTCTCGGGAAAGCCCACCAACCGGCCTGGTTGTCTTCGGTGGCGGTACCAAGCTGACCGTCCTAGGTCAACCCAAGGCTGCCCCCAGCGTGACCCTGTTCCCCCCCAGCAGCAAGAAACTGCAGGCCAACAAGGCCACCCTGGTCTGCCTGATCAGCGACTTCTACCCAGGCGCCGTGACCGTGGCCTGGAAGGCCGACAGCAGCCCCGTGAAGGCCGGCGTGGAGACCACCACCCCCAGCAAGCAGAGCAACAACAAGTACGCCGCCAGCAGCTACCTGAGCCTGACCCCCGAGCAGTGGAAGAGCCACAGGTCCTACAGCTGCCAGGTGACCCACGAGGGCAGCACCGTGGAGAAAACCGTGGCCCCCACCGAGTGCAGC 9C7 VHCAGGTGCAATTGGTTCAATCTGGTGCTGAAGTAAAAAAACCGGGCGCTTCCGTT 295AAAGTGAGCTGCAAAGCATCCGGATACACCTTCACTTCCTATTACATGCACTGGGTTCGTCAAGCCCCGGGCCAGGGTCTGGAATGGATGGGCATCATTAACCCAAGCGGTGGCTCTACCTCCTACGCGCAGAAATTCCAGGGTCGCGTCACGATGACCCGTGACACTAGCACCTCTACCGTTTATATGGAGCTGTCCAGCCTGCGTTCTGAAGATACTGCAGTGTACTACTGTGCACGCGGTGACTGGTCTTACTACATGGACTATTGGGGTCAAGGCACCCTCGTAACGGTTTCTTCT 9C7 VLGATATTGTTATGACTCAATCTCCACTGTCTCTGCCGGTGACTCCAGGCGAACCG 296GCGAGCATTTCTTGCCGTTCCAGCCAGTCTCTGCTGCACTCCAACGGCTACAACTATCTCGATTGGTACCTGCAAAAACCGGGTCAGAGCCCTCAGCTGCTGATCTACCTGGGCTCTAACCGCGCTTCCGGTGTACCGGACCGTTTCAGCGGCTCTGGATCCGGCACCGATTTCACGTTGAAAATCAGCCGTGTTGAAGCAGAAGACGTGGGCGTTTATTACTGTATGCAGGCACGGCAGACCCCAACTTTTGGTCAAGGCACCAAGGTC GAAATTAAA9C7[EE]_ CAGGTGCAATTGGTTCAATCTGGTGCTGAAGTAAAAAAACCGGGCGCTTCCGTT 297CD3[VLCH1]_ AAAGTGAGCTGCAAAGCATCCGGATACACCTTCACTTCCTATTACATGCACTGG Fc-GTTCGTCAAGCCCCGGGCCAGGGTCTGGAATGGATGGGCATCATTAACCCAAGC knob_GGTGGCTCTACCTCCTACGCGCAGAAATTCCAGGGTCGCGTCACGATGACCCGT PGLALAGACACTAGCACCTCTACCGTTTATATGGAGCTGTCCAGCCTGCGTTCTGAAGAT pETR14974ACTGCAGTGTACTACTGTGCACGCGGTGACTGGTCTTACTACATGGACTATTGGGGTCAAGGCACCCTCGTAACGGTTTCTTCTGCTAGCACCAAGGGCCCCTCCGTGTTTCCTCTGGCCCCTTCCAGCAAGTCCACCTCTGGCGGAACTGCCGCTCTGGGCTGCCTGGTGGAAGATTACTTCCCCGAGCCCGTGACCGTGTCCTGGAATTCTGGCGCTCTGACCTCCGGCGTGCACACCTTTCCAGCTGTGCTGCAGTCCTCCGGCCTGTACTCCCTGTCCTCCGTCGTGACAGTGCCCTCCAGCTCTCTGGGCACCCAGACCTACATCTGCAACGTGAACCACAAGCCCTCCAACACCAAGGTGGACGAGAAGGTGGAACCCAAGTCCTGCGACGGTGGCGGAGGTTCCGGAGGCGGAGGATCCCAGGCTGTCGTGACCCAGGAACCCTCCCTGACAGTGTCTCCTGGCGGCACCGTGACCCTGACCTGTGGATCTTCTACCGGCGCTGTGACCACCTCCAACTACGCCAATTGGGTGCAGGAAAAGCCCGGCCAGGCCTTCAGAGGACTGATCGGCGGCACCAACAAGAGAGCCCCTGGCACCCCTGCCAGATTCTCCGGTTCTCTGCTGGGCGGCAAGGCTGCCCTGACTCTGTCTGGTGCTCAGCCTGAGGACGAGGCCGAGTACTACTGCGCCCTGTGGTACTCCAACCTGTGGGTGTTCGGCGGAGGCACCAAGCTGACCGTGCTGTCCAGCGCTTCCACCAAGGGACCCAGTGTGTTCCCCCTGGCCCCCAGCTCCAAGTCTACATCCGGTGGCACAGCTGCCCTGGGATGTCTCGTGAAGGACTACTTTCCTGAGCCTGTGACAGTGTCTTGGAACAGCGGAGCCCTGACCAGCGGAGTGCACACATTCCCTGCAGTGCTGCAGAGCAGCGGCCTGTATAGCCTGAGCAGCGTCGTGACCGTGCCTTCCTCTAGCCTGGGAACACAGACATATATCTGTAATGTGAATCATAAGCCCAGTAATACCAAAGTGGATAAGAAAGTGGAACCTAAGAGCTGCGATAAGACCCACACCTGTCCCCCCTGCCCTGCTCCTGAAGCTGCTGGTGGCCCTAGCGTGTTCCTGTTCCCCCCAAAGCCCAAGGACACCCTGATGATCTCCCGGACCCCCGAAGTGACCTGCGTGGTGGTGGATGTGTCCCACGAGGACCCTGAAGTGAAGTTCAATTGGTACGTGGACGGCGTGGAAGTGCACAACGCCAAGACCAAGCCTAGAGAGGAACAGTACAACTCCACCTACCGGGTGGTGTCCGTGCTGACAGTGCTGCACCAGGACTGGCTGAACGGCAAAGAGTACAAGTGCAAGGTGTCCAACAAGGCCCTGGGCGCTCCCATCGAAAAGACCATCTCCAAGGCCAAGGGCCAGCCCCGGGAACCCCAGGTGTACACCCTGCCCCCATGCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTGTGGTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAA 9C7[EE]_CAGGTGCAATTGGTTCAATCTGGTGCTGAAGTAAAAAAACCGGGCGCTTCCGTT 298 Fc-AAAGTGAGCTGCAAAGCATCCGGATACACCTTCACTTCCTATTACATGCACTGG hole_GTTCGTCAAGCCCCGGGCCAGGGTCTGGAATGGATGGGCATCATTAACCCAAGC PGLALAGGTGGCTCTACCTCCTACGCGCAGAAATTCCAGGGTCGCGTCACGATGACCCGT pETR14975GACACTAGCACCTCTACCGTTTATATGGAGCTGTCCAGCCTGCGTTCTGAAGATACTGCAGTGTACTACTGTGCACGCGGTGACTGGTCTTACTACATGGACTATTGGGGTCAAGGCACCCTCGTAACGGTTTCTTCTGCTAGCACCAAGGGCCCCTCCGTGTTCCCCCTGGCCCCCAGCAGCAAGAGCACCAGCGGCGGCACAGCCGCTCTGGGCTGCCTGGTCGAGGACTACTTCCCCGAGCCCGTGACCGTGTCCTGGAACAGCGGAGCCCTGACCTCCGGCGTGCACACCTTCCCCGCCGTGCTGCAGAGTTCTGGCCTGTATAGCCTGAGCAGCGTGGTCACCGTGCCTTCTAGCAGCCTGGGCACCCAGACCTACATCTGCAACGTGAACCACAAGCCCAGCAACACCAAGGTGGACGAGAAGGTGGAGCCCAAGAGCTGCGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAAGCTGCAGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCGGCGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTGCACCCTGCCCCCATCCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTCTCGTGCGCAGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCGTGAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAA 9C7 LCGATATTGTTATGACTCAATCTCCACTGTCTCTGCCGGTGACTCCAGGCGAACCG 299 [RK]GCGAGCATTTCTTGCCGTTCCAGCCAGTCTCTGCTGCACTCCAACGGCTACAAC pETR14980TATCTCGATTGGTACCTGCAAAAACCGGGTCAGAGCCCTCAGCTGCTGATCTACCTGGGCTCTAACCGCGCTTCCGGTGTACCGGACCGTTTCAGCGGCTCTGGATCCGGCACCGATTTCACGTTGAAAATCAGCCGTGTTGAAGCAGAAGACGTGGGCGTTTATTACTGTATGCAGGCACGGCAGACCCCAACTTTTGGTCAAGGCACCAAGGTCGAAATTAAACGTACGGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATCGGAAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGA GAGTGT

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity ofunderstanding, the descriptions and examples should not be construed aslimiting the scope of the invention. The disclosures of all patent andscientific literature cited herein are expressly incorporated in theirentirety by reference.

1. A T cell activating bispecific antigen binding molecule comprising(i) a first antigen binding moiety which is a Fab molecule capable ofspecific binding to CD3, and which comprises at least one heavy chaincomplementarity determining region (CDR) amino acid sequence selectedfrom the group consisting of SEQ ID NO: 37, SEQ ID NO: 38 and SEQ ID NO:39 and at least one light chain CDR selected from the group of SEQ IDNO: 32, SEQ ID NO: 33, SEQ ID NO: 34; (ii) a second antigen bindingmoiety capable of specific binding to Folate Receptor 1 (FolR1).
 2. TheT cell activating bispecific antigen binding molecule of claim 1,wherein the first antigen binding moiety comprises a variable heavychain comprising an amino acid sequence of SEQ ID NO: 36 and a variablelight chain comprising an amino acid sequence of SEQ ID NO:
 31. 3. The Tcell activating bispecific antigen binding molecule of claim 1 or 2,additionally comprising (i) a third antigen binding moiety capable ofspecific binding to FolR1.
 4. The T cell activating bispecific antigenbinding molecule of claim 3, wherein the second and third antigenbinding moiety capable of specific binding to FolR1 comprise identicalheavy chain complementarity determining region (CDR) and light chain CDRsequences.
 5. The T cell activating bispecific antigen binding moleculeof claim 4, wherein the third antigen binding moiety is identical to thesecond antigen binding moiety.
 6. The T cell activating bispecificantigen binding molecule of claim 3, wherein at least one of the secondand third antigen binding moiety is a Fab molecule.
 7. The T cellactivating bispecific antigen binding molecule of claim 1, additionallycomprising (i) an Fc domain composed of a first and a second subunitcapable of stable association.
 8. The T cell activating bispecificantigen binding molecule of claim 7, wherein the first antigen bindingmoiety and the second antigen binding moiety are each connected at theC-terminus of the Fab heavy chain to the N-terminus of the first orsecond subunit of the Fc domain.
 9. The T cell activating bispecificantigen binding molecule of claim 7 or 8, wherein a third antigenbinding moiety is connected at the C-terminus of the Fab heavy chain tothe N-terminus of the Fab heavy chain of the first antigen bindingmoiety via a peptide linker. 10.-21. (canceled)
 22. The T cellactivating bispecific antigen binding molecule of claim 1, which bindsto a human FolR1, a cynomolgus monkey FolR1 and a murine FolR1.
 23. TheT cell activating bispecific antigen binding molecule of claim 1, whichbinds to a human FolR1 and a cynomolgus monkey FolR1 and not a murineFolR1.
 24. The T cell activating bispecific antigen binding molecule ofclaim 1, wherein the first antigen binding moiety is a crossover Fabmolecule wherein either the variable or the constant regions of the Fablight chain and the Fab heavy chain are exchanged.
 25. The T cellactivating bispecific antigen binding molecule of claim 1, comprisingnot more than one antigen binding moiety capable of specific binding toCD3.
 26. The T cell activating bispecific antigen binding molecule ofclaim 7, wherein the first and the second antigen binding moiety and theFc domain are part of an immunoglobulin molecule.
 27. The T cellactivating bispecific antigen binding molecule of claim 26, wherein theFc domain is an IgG class immunoglobulin.
 28. The T cell activatingbispecific antigen binding molecule of claim 27, wherein the Fc domainis a human IgG₁ or IgG₄ Fc domain.
 29. The T cell activating bispecificantigen binding molecule of claim 7 or 28, wherein the Fc domaincomprises a modification promoting the association of the first and thesecond subunit of the Fc domain.
 30. The T cell activating bispecificantigen binding molecule of claim 29, wherein in the CH3 domain of thefirst subunit of the Fc domain an amino acid residue is replaced with anamino acid residue having a larger side chain volume, thereby generatinga protuberance within the CH3 domain of the first subunit which ispositionable in a cavity within the CH3 domain of the second subunit,and in the CH3 domain of the second subunit of the Fc domain an aminoacid residue is replaced with an amino acid residue having a smallerside chain volume, thereby generating a cavity within the CH3 domain ofthe second subunit within which the protuberance within the CH3 domainof the first subunit is positionable.
 31. The T cell activatingbispecific antigen binding molecule of claim 7, wherein the Fc domaincomprises at least one amino acid substitution that reduces binding toan Fc receptor and/or effector function, as compared to a native IgG₁ Fcdomain.
 32. The T cell activating bispecific antigen binding molecule ofclaim 31, wherein each subunit of the Fc domain comprises three aminoacid substitutions that reduce binding to an activating Fc receptorand/or effector function wherein said amino acid substitutions areL234A, L235A and P329G (Kabat numbering).
 33. (canceled)
 34. The T cellactivating bispecific antigen binding molecule of claim 31, wherein theeffector function is antibody-dependent cell-mediated cytotoxicity(ADCC).
 35. The T cell activating bispecific antigen binding molecule ofclaim 1, wherein the T cell activating bispecific antigen bindingmolecule induces at least one of (a) proliferation of a human CD3positive T cell in vitro; (b) human peripheral blood mononuclear cellmediated killing of a FolR1-expressing human tumor cell in vitro; (c) Tcell-mediated killing of a FolR1-expressing human tumor cell in vitro;and (d) upregulation of cell surface expression of at least one of CD25and CD69 on the T cell as measured by flow cytometry. 36.-37. (canceled)38. The T cell activating bispecific antigen binding molecule of claim35, wherein the FolR1-expressing human tumor cell is a Hela, Skov-3, ora cell.
 39. The T cell activating bispecific antigen binding molecule ofclaim 35 or 38, wherein the T cell activating bispecific antigen bindingmolecule induces T cell mediated killing of the FolR1-expressing humantumor cell in vitro with an EC50 of between about 36 pM and about 39573pM after 24 hours.
 40. The T cell activating bispecific antigen bindingmolecule of claim 39, wherein the T cell activating bispecific antigenbinding molecule induces T cell mediated killing of the FolR1-expressingtumor cell in vitro with an EC50 of about 36 pM after 24 hours.
 41. TheT cell activating bispecific antigen binding molecule of claim 39,wherein the T cell activating bispecific antigen binding moleculeinduces T cell mediated killing of the FolR1-expressing tumor cell invitro with an EC50 of about 178.4 pM after 24 hours.
 42. The T cellactivating bispecific antigen binding molecule of claim 39, wherein theT cell activating bispecific antigen binding molecule induces T cellmediated killing of the FolR1-expressing tumor cell in vitro with anEC50 of about 134.5 pM or greater after 48 hours.
 43. (canceled)
 44. TheT cell activating bispecific antigen binding molecule of claim 35,wherein the T cell is a CD4 positive T cell or a CD8 positive T cell.45. The T cell activating bispecific antigen binding molecule of claim1, wherein the T cell activating bispecific antigen binding moleculebinds human FolR1 with an apparent K_(D) of about 5.36 pM to about 4 nM.46. The T cell activating bispecific antigen binding molecule of claim1, wherein the T cell activating bispecific antigen binding moleculebinds human and cynomolgus FolR1 with an apparent K_(D) of about 4 nM.47. (canceled)
 48. The T cell activating bispecific antigen bindingmolecule of claim 1, wherein the T cell activating bispecific antigenbinding molecule binds human FolR1 with a monovalent binding K_(D) of atleast about 1000 nM.
 49. (canceled)
 50. The T cell activating bispecificantigen binding molecule of claim 1, wherein the T cell activatingbispecific antigen binding molecule binds to a conformational epitope ofa human FolR1 expressed on a human tumor cell.
 51. The T cell activatingbispecific antigen binding molecule of claim 1, wherein the T cellactivating bispecific antigen binding molecule does not bind to humanFolate Receptor 2 (FolR2) or to human Folate Receptor 3 (FolR3).
 52. TheT cell activating bispecific antigen binding molecule of claim 1,wherein the antigen binding moiety binds to a FolR1 polypeptidecomprising the amino acids 25 to 234 of human FolR1 (SEQ ID NO:227). 53.The T cell activating bispecific antigen binding molecule of claim 1,wherein the FolR1 antigen binding moiety binds to a FolR1 polypeptidecomprising the amino acid sequence of SEQ ID NOs:227, 230 and 231, andwherein the FolR1 antigen binding moiety does not bind to a FolRpolypeptide comprising the amino acid sequence of SEQ ID NOs:228 and229. 54.-103. (canceled)
 104. An isolated polynucleotide encoding the Tcell activating bispecific antigen binding molecule of claim 1 or afragment thereof. 105.-109. (canceled)
 110. A polypeptide encoded by thepolynucleotide of claim
 104. 111. A vector comprising the polynucleotideof claim
 104. 112. A host cell comprising the polynucleotide of claim104 or the vector of claim
 111. 113. A method of producing the T cellactivating bispecific antigen binding molecule capable of specificbinding to CD3 and a target cell antigen, comprising the steps of a)culturing the host cell of claim 112 under conditions suitable for theexpression of the T cell activating bispecific antigen binding moleculeand b) recovering the T cell activating bispecific antigen bindingmolecule.
 114. A T cell activating bispecific antigen binding moleculeproduced by the method of claim
 113. 115. A pharmaceutical compositioncomprising the T cell activating bispecific antigen binding molecule ofclaim 1 and a pharmaceutically acceptable carrier. 116.-119. (canceled)120. A method of treating a disease in an individual, comprisingadministering to said individual a therapeutically effective amount of acomposition comprising the T cell activating bispecific antigen bindingmolecule of claim 1 in a pharmaceutically acceptable form.
 121. Themethod of claim 120, wherein said disease is cancer.
 122. A method forinducing lysis of a FolR1⁺ target cell, comprising contacting a targetcell with the T cell activating bispecific antigen binding molecule ofclaim 1 in the presence of a CD3⁺ T cell.
 123. (canceled)